SUPPLEMENTARY FIGURE

Figure S1. E. coli K1 promotes reciprocal regulation of Ecgp96/AT1R and PPAR-γ/GLUT-

1 levels in the hippocampus of newborn mice. The brain sections were stained with tomato

lectin (green) to visualize microvessels and for (A) Ecgp96, AT1R, (B) PPAR-γ, and GLUT-1

(red) expression in the hippocampus. DAPI (blue) was used to stain the nuclei. Scale bar = 20

µm.

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SUPPLEMENTARY METHODS

Bacterial strains. The prototype Escherichia coli K1 strain (O18:K1:H7) - a spontaneous

rifampicin-resistant mutant of strain RS218 - and its ompA deletion mutant were described

previously. [27]

Chemicals and reagents. Telmisartan (TS) (Cat. No. 5139), Rosiglitazone (RG) (Cat. No.

5325), GW1929 (Cat. No. 1664), GW9662 (Cat. No. 1508), CP 775146 (Cat. No. 4190), and L-

165,041 (Cat. No. 1856) were purchased from Tocris Bioscience, Minneapolis, MN. Antibodies

to Ecgp96 (GRP94) (Cat. No. GTX103203) and β-actin (Cat. No. GTX109639) were purchased

from GeneTex, Irvine, CA. Antibodies to GLUT-1 (Cat. No. sc-7903) and AT1R (Cat. No. sc-

1173-G) were purchased from Santa Cruz Biotechnology, Dallas, TX. Antibodies to PPAR-γ

(bs-4590R) were purchased from Bioss Antibodies, Woburn, MA. Antibodies to PPAR-α (Cat.

No. bs-3614R), PPAR-β (Cat. No. bs-0250R), GLUT-3 (bs-1207R), and GLUT-4 (bs-0384R)

were purchased from One World Lab, San Diego, CA. Plasmids over-expressing PPAR-γ (Cat.

No. 8895), GLUT-1 (Cat. No. 18085), and GLUT-4 (Cat. No. 18087) were purchased from

Addgene, Cambridge, MA.

Invasion assays. Primary HBMECs were cultured to a density of 105 cells per well in 24-well

plates. Bacteria were added at a multiplicity of infection (MOI) of 100 for 90 minutes at 37°C

with 5% CO2. The cells were further incubated with medium containing 50 µg gentamicin for 1

hour. The cells were lysed with 0.5% Triton X-100, and then intracellular bacteria recovered

were plated on blood agar. Percentage invasion was determined by comparing each strain's

recovered bacteria to the input. HBMECs were pre-treated with the respective compounds for 1

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hour prior to infection: TS (39 µM), RG (50 µM), GW1929 (50 µM), GW9662 (33 nM), CP

775146 (24.5 nM), L-165,041 (125 nM). For transfection experiments, HBMECs were

transfected with plasmids expressing PPAR-γ (FL-PPAR-γ), GLUT-1 (FL-GLUT-1), or GLUT-4

(FL-GLUT-4) using Lipofectamine 3000 (ThermoFisher Scientific, Waltham, MA, Cat. No.

L3000008) for 24 hours prior to performing invasion assays, flow cytometry analysis, or

Western blots.

Western blots. HBMECs were infected as described previously [27], and 30 µg of whole cell

lysate was run on SDS-PAGE and subjected to immunoblotting with the antibodies listed above.

For compound treatment and plasmid expression, HBMECs were treated or transfected as

described previously, infected with E. coli K1, lysed, and subjected to Western blot analysis.

Flow cytometry. HBMECs were infected as described previously [27], and subjected to flow

cytometry analyses with antibodies to PPAR-γ, GLUT-1, and GLUT-4. For overexpression

analysis, plasmids were transfected as described previously. Staining with secondary antibody

alone was used as an isotype control. At least 10,000 cells per sample were analyzed with a BD

FACS Calibur flow cytometer.

Glucose measurement. HBMECs were infected, treated, and/or transfected as described

previously. After killing extracellular bacteria with 50 µg gentamicin, the cells were washed

three times with PBS, and incubated with 100 µl assay buffer (Biovision, Milpitas, CA, Cat. No.

K606-100) to lyse the cells. From that suspension, 50 µl was transferred to a 96-well plate, and

an equal volume of the reaction mix was added to all wells, including the glucose standards. The

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plate was incubated for 30 minutes at 37°C in the dark, and the glucose uptake was measured at

550 nm.

Trans-endothelial electrical resistance (TEER) measurement. HBMECs were infected,

treated, and/or transfected as previously described. Bacteria was added at time = 0 min. TEER

measurements were taken at 15, 30, 60, 90, and 120 min post-infection using a Millicell ERS

microhmmeter (Millipore, Bedford, MA, Cat. No. MERS00001).

Mouse infections and brain histology. Animal experiments were carried out according to

guidelines mandated by the Office of Laboratory Animal Welfare at NIH. Protocols were

approved by the Institutional Animal Care and Use Committee of Children's Hospital of Los

Angeles (Protocol #59-14). Briefly, 2-day old C57BL/6 pups were infected with 100 colony

forming units of E. coli K1 through intraperitoneal (IP) route. The pups were monitored up to 24

hours post-infection for signs of illness. Pups were sacrificed, and CSF, blood, and brain

samples were collected and used as required for bacterial growth, enumeration, cytokine ELISA,

brain sectioning, and immunostaining. For treatment studies, the pups were treated with

Telmisartan (TS) or Rosiglitazone (RG) 6 h prior to infection, during infection, and 18 h post-

infection. Hematoxylin and Eosin (H&E), immunofluorescence, and brain microvessel staining

were executed as reported before [14, 24, 26].

ELISA. Cytokine ELISA was performed according to the ProcartaPlex Mouse Immunoassay

Manual (eBioscience, San Diego, CA) using Mouse Th1/Th2 Essential 6Plex (Cat. No. EPX060-

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20831-SA), Mouse IL-10 Simplex (Cat. No. EPX010-20614-901), and Mouse IL-1β Simplex

(Cat. No. EPX010-26002-901). Samples were analyzed on a Luminex-200 analyzer.

Confocal microscopy. Paraffin-embedded brain sections were sequentially rehydrated using

xylene, 100% ethanol, 95% ethanol, 70% ethanol, and water. Antigen unmasking was performed

by incubating the sections in 0.25% trypsin-EDTA at 37°C for 20 min, followed by a DI water

wash. The sections were blocked in blocking buffer (sterile PBS, 0.5% Triton X-100, 3%

normal goat serum) at room temperature for 30 min. The slides were washed again in DI water,

and respective antibodies to Ecgp96, AT1R, PPAR-γ, and GLUT-1 in PBS/0.5% Triton X-100

containing 1% BSA were added to respective sections and incubated overnight at 4°C. For

microvessel staining, 10 mg/ml tomato lectin (DyLight 594 Lycopersicon esculentum, Vector

Laboratories, Burlingame, CA, Cat. No. DL-1177) was added along with primary antibodies.

The sections were incubated with Alexa 647-conjugated secondary antibodies (ThermoFisher

Scientific, Waltham, MA, Cat. No. A21244) at room temperature for 1 hour along with DAPI.

Images were obtained on a Zeiss LSM 700 confocal microscope (Carl Zeiss Microscopy)

controlled by Micro-Manager software at 40X with multiple fields of view.

Statistical analysis. The unpaired Student’s t-test was used for statistical analysis for all the

experiments. Furthermore, one-way ANOVA was used for statistical analysis of blood and brain

bacterial counts from newborn mice. P-values < 0.05 were considered statistically significant.

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