Serum Amyloid A Induction by UPEC Figure 3. ELISA of mouse sera at different time points after infection. Four mice were inoculated with UTI89 via either (A) transurethral catheterization or (B) IP injection. (A) Serum levels of SAA1/2 and SAA3 in mice were quantified by ELISA in sham inoculated animals and at 1, 4, and 14 d after UTI89 instillation into the bladder (UTI). SAA3 was not detected in these samples. (B) Substantially higher serum levels of SAA1/2 and SAA3 were detected 1 d after infection with UTI89 via IP injection. *, p,0.05 relative to sham inoculated animals as determined by Student’s t test. The mean +/2 SD of triplicate experiments are shown. doi:10.1371/journal.pone.0032933.g003 USA) and Power SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) using SAA-specific primers and ribosomal protein L32 primers serving as endogenous control for normalization as described in [25]. Dissociation curves of products showed only one peak in each PCR reaction. 900 nM of forward and reverse primers were used to amplify SAA1/2 and SAA3, while 300 nM of primers were used for L32. Data analysis was done using 2DDCt method with normalization to the level of L32. Biofilm and Bacterial Growth Assays Microtiter plate-based biofilm assays were carried out as previously described [2]. In brief, bacterial strains from overnight shaking cultures were diluted 1:100 in M9 minimal medium, and quadruplicate 100-ml samples were incubated 60, 0.5, 1 and 5 mg/ml SAA in 96-well pinchbar flat-bottomed polystyrene microtiter plates with lids for two days at 30uC without shaking. Nonadherent bacterial cells were then removed by gentle washings with distilled H2O and the remaining biofilm-associated bacteria were stained for 10 min at room temperature with 150 ml of a 0.1% solution of crystal violet in water, (Sigma-Aldrich, MO, USA). Wells were then washed with H2O, air dried for 30 min, and incubated for 15 min with 200 ml dimethyl sulfoxide (DMSO) to release the incorporated dye. After transferring a 150-ml aliquot from each well to a new microtiter plate, A562 was measured using a Synergy HT multidetection plate reader (BioTek Instruments, Inc., VT, USA). Bacterial growth curves were acquired using quadruplicate 200- ml cultures of MG1655, UTI89, and F11 shaking in M9 minimal medium 60, 5, or 20 mg/ml SAA at 37uC in 100-well honeycomb plates using a Bioscreen C instrument (Growth Curves, USA), as previosuly described [32]. Results To investigate the ability of UPEC to affect SAA expression in the host, we employed a well-established mouse UTI model system. At 1 and 4 d following catheterization of adult female C57BL/6JOLaHsd mice with the UPEC isolate UTI89, levels of SAA1/2 in the urothelium were observed to be notably elevated relative to uninfected controls (Fig. 1A–C). Within the urothelial cells, SAA1/2 was primarily situated within the cytosol, where it co-localized strongly with internalized UPEC (Fig. 1D and E). Upregulation of SAA at day 1 post-inoculation of the bladder with UTI89 was confirmed by qRT-PCR of the bladder wall as well as the isolated urothelium and the liver (Fig. 2). SAA1/2 expression levels were elevated more than 4.8-fold in the liver and urothelium, and more than 30-fold in the bladder wall, while the relative induction of SAA3 expression in these tissues in response to UTI was less robust. However, in control experiments in which mice were infected systemically with UTI89 delivered via intraperitoneal (IP) injection, SAA3 expression was markedly increased in the liver (more than 300-fold) as well as in the urothelium (,20-fold) and bladder wall (,90-fold). Within 1 d following instillation of mouse bladders with UTI89, we observed by ELISA a substantial 8-fold increase in SAA1 serum levels, which decreased by day 4 and remained subdued up to day 14 (Fig. 3A). In contrast, serum levels of SAA3 were not altered in response to UTI (data not shown). However, following IP injection of UTI89 we did detect significant and notably large increases in circulating SAA3 levels along with corresponding, although smaller, increases in SAA1/2 levels (Fig. 3B). This is the first time, to our knowledge, that serum SAA3 protein levels have been shown to increase substantially in response to UPEC delivered intraperitoneally. Previous work indicated that recombinant SAA1/2 expressed by cultured host cell lines can inhibit the growth and viability of laboratory E. coli K-12 strains [12], raising the possibility that SAA may act similarly against UPEC isolates during the course of a UTI. However, in in vitro assays, we found that growth of the UPEC isolates UTI89 and F11 in M9 medium was unfazed by SAA concentrations up to 100 mg/ml (data not shown and Fig. 4), indicating that SAA does not have direct bactericidal or bacteriostatic effects on UPEC. Interestingly, SAA also had no effect on growth of the reference K-12 isolate MG1655 in our assays. In considering alternate means by which SAA could potentially affect the pathogenesis of UTIs, we noted previous reports indicating that SAA can opsonize Gram-negative bacteria by binding the abundant outer membrane protein OmpA [4,9]. OmpA is an important facilitator of biofilm formation by UPEC and other E. coli isolates (unpublished observations and [15]), and biofilm formation both on and within the urothelium is thought to promote the establishment and persistence of UPEC within the urinary tract [1,30]. To test possible effects of SAA on biofilm formation by UPEC, we employed standard in vitro microtiter plate PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e32933