All animal work was conducted according to relevant U.S. and international guidelines. Specifically, animal experimental work was reviewed and approved as protocol 14-03 by the Institutional Animal Care and Use Committee of the Biomedical Research Institute (Rockville, Maryland, USA). Our Institutional Animal Care and Use Committee guidelines comply with the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals.
Six to seven-week-old male and female BALB/c mice (Charles River Laboratories, Wilmington, MA, USA) were housed under 12 h light- dark cycles in temperature-controlled holding rooms with unlimited access to dry mouse chow and water. Newly received mice were acclimated to the animal facility for at least one week prior to experimental use.
IPSE protein production
Recombinant H06 H-IPSE (one of the major Schistosoma haematobium IPSE orthologs) and an NLS mutant of H06 H-IPSE (the wild type NLS sequence SKRGRKY changed to SAAGAAY) were produced in HEK293-6E cells as previously described .
One day prior to UTI induction, mice underwent tail vein injection with phosphate-buffered saline or 25 mg of H06 H-IPSE (or its NLS mutant) in phosphate-buffered saline.
UTI89 infection of mice
The uropathogenic Escherichia coli (UPEC) strain UTI89 was derived from a patient with cystitis . Briefly, UTI89 was streaked on an LB agar plate to obtain a single colony which was then inoculated into 10 ml of LB broth and grown at 37°C overnight under static conditions to promote type 1-pilus expression[32,33]. Bacteria were then subcultured 1:100 into 10 ml of fresh medium, followed by growth at 37°C for another 18 h. These cultures were centrifuged for 10 min at 14,000 rpm, resuspended in 2 ml PBS, and then diluted to approximately 2 x 109 CFU per ml as estimated using a spectrophotometer (OD600 nm). 50 μl of this suspension (108 CFU) was inoculated into the bladders of female mice by transurethral catheterization . Male mice were not tested for UTI susceptibility; catheterization of male mice is technically very challenging.
Bacterial titer determinations
Freshly collected urine was serially diluted 100- and 1,000-fold in sterile PBS. Twenty-five microliters of each dilution was plated onto MacConkey agar plates. CFU were counted after overnight incubation at 37°C. The limits of detection were 4,000 CFU/ml for urine.
Bladder tissues were fixed in neutral buffered formalin, dehydrated, and embedded in paraffin. Five-micrometer sections were stained with hematoxylin and eosin. Histology was analyzed for each bladder by a board-certified pathologist (JIO) in a blinded fashion, using the following scoring system:
1 urothelial hyperplasia with reactive nuclear atypia and architectural disarray
2 segmental urothelial ulceration
1 perivascular edema with expansion but preservation of bladder architecture
2 severe edema with effacement of the submucosa
1 perivascular acute inflammatory infiltrates
2 frank acute inflammation of the urothelium with urothelial microabscesses
1 sparse hemorrhage
2 frank intra-tissue hemorrhage
0 Normal papillae
1 blunted/wide contraction papillae
2 effaced contraction papillae
0.5 gradations for focality
RNA was isolated from mouse bladders using TRIzol Reagent and PureLink RNA Mini Kit (Invitrogen), according to manufacturers’ instructions. Briefly, aseptically excised bladders were homogenized in 1 ml TRIzol Reagent by bead-beating using ceramic beads (Omni International) and a mini-bead beater (Biospec). Following a 5-min incubation, 0.2 ml chloroform was added and again incubated for 3 min before centrifugation at 12,000 × g for 15 min to separate homogenates into aqueous and organic phases. The aqueous supernatant (~400μl) was mixed with an equal volume of 70% ethanol before binding the mixture to RNA binding columns by centrifugation. On-column DNase digestion (Invitrogen) was performed for 30 minutes, following the manufacturer’s protocols. After column washes and drying, RNA was eluted in RNase-free water, quantified and its quality checked using a NanoDrop 1000 spectrophotometer (Thermo Scientific) and Bioanalyzer 2100 (Agilent).
cDNA synthesis and real-time PCR
cDNA synthesis was performed using the RT2 First Strand cDNA kit (SABiosciences). Real-time PCR was performed for anti-microbial peptide gene expression using an Mx3005p thermocycler (Stratagene) using an RT2 custom PCR array (SABiosciences) with RT2 SYBR green quantitative PCR (qPCR) master mixes (SABiosciences). Cycle thresholds (CT) were calculated for each reaction. Using the comparative CT method, relative gene expression was calculated as 2(-DDCT), where DCT = CT (gene of interest) - DCT ( b- actin). DDCT was calculated as DCT (H-IPSE injection and bacterially infected) - DCT (bacterially infected). Data are expressed as means ± standard deviations (SDs). P values were calculated using Mann-Whitney U tests comparing DCT of the groups receiving H-IPSE and UTI challenge to the groups receiving UTI challenge alone (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Melt curves confirmed the specificity of PCR reactions. PCR primer sequences are shown in Supplemental Table 1.
Toxicity assays were performed using four groups including one vehicle control (phosphate-buffered saline) and three H-IPSE treated groups (0.5, 1, and 2 mg/kg/day via tail vein injection daily for 7 days), using BALB/c mice. The dosing volume was kept constant at 6.35 mL/kg/day for each mouse. Parameters evaluated included clinical signs of illness, body weights, percent body weight gain, feed consumption, hematology, clinical chemistry, and after 7 days, organ weights, gross pathology, and histopathology.
Two groups of mice (BALB/c 6-week old, n = 4 in each group) were injected with either PBS or 25μg H-IPSE intravenously. Mice were bled 5 minutes after injection with PBS or H-IPSE via cheek bleed and 30 minutes post injection via terminal heart bleed. Sera was collected and immediately used for histamine analysis using a mouse histamine ELISA kit from MyBioSource using manufacturer’s instructions (Catalog # MBS725193).
Statistical analyses were performed using GraphPad software. Except where noted otherwise, Mann-Whitney U tests and Student t tests were used to evaluate statistical significance for nonparametrically and parametrically distributed data, respectively. ANOVA with Tukey’s multiple comparison test were used for comparisons of three or more experimental groups. P values of <0.05 were defined as significant.