Streptococcus agalactiae disrupts P-glycoprotein function in brain endothelial cells

We first sought to determine if P-gp function was altered during infection. To do so, we utilized induced pluripotent stem cell (iPSC)-derived BEC-like cells that have been shown to possess P-gp activity [21‐23]. iPSC-derived BECs were differentiated and express expected endothelial markers as previously described (Additional file 1: Figure S1A-F) [21‐23]. Using a substrate accumulation assay and consistent with prior observations, BECs treated with the P-gp inhibitor Cyclosporine A (CsA) accumulated more of the P-gp substrate Rhodamine 123 (R123) than non-CsA-treated cells, indicating that P-gp is active in these BECs. Following GBS infection, we observed a significant increase of R123 accumulation in BECs when compared to uninfected BECs, to levels matching those with CsA inhibition (Fig. 1a). Addition of CsA to the infected condition made no impact on accumulation, and the combined data suggest P-gp function is diminished during GBS infection. To determine if the observation was substrate specific, similar experiments were performed with a different P-gp substrate, FLUO-3-AM, and a similar increase in substrate accumulation in GBS-infected BECs was observed (Fig. 1b). In addition, inhibition of P-gp with the second generation, more specific inhibitor PSC-833 [6] yielded similar results to CsA inhibition (Fig. 1c). To determine if this impact on P-gp function is specific to meningeal pathogens, P-gp function was assayed following incubation with a genetically similar non-pathogenic bacterium, Lactococcus lactis. In contrast to GBS effects, L. lactis did not inhibit P-gp function in an R123 accumulation assay (Fig. 1d). We and others have previously shown that inhibition of BCRP or MRP family proteins with Ko143 or MK571 respectively, in iPSC-BECs resulted in functional inhibition of those transporters [21, 22, 24‐29]. Inhibition of BCRP or MRPs using Ko143 or MK571, did not result in an increase in R123 accumulation (Fig. 1e). This suggests that R123 efflux, in our model, is primarily mediated by P-gp and that accumulation can be increased by CsA. Taken together, these observations suggest that GBS infection results in reduced P-gp function in BECs as measured by substrate accumulation.

To ensure that the observed decrease in P-gp was not a function of the iPSC origin of BECs, we also examined P-gp function in a human P-gp-overexpressing Madin-Darby canine kidney (MDCK) cell line [30]. As a result of the overexpression of human P-gp, there is a greater relative increase of R123 accumulation upon CsA inhibition (Fig. 2a). Despite this increase in P-gp activity, GBS-mediated P-gp inhibition could still be observed with complete inhibition of P-gp activity at high multiplicity of infection (MOI) (Fig. 2a). These data suggest that P-gp inhibition is related to the balance of P-gp expression and level of bacterial interaction, and that GBS-mediated P-gp inhibition may not be specific to BECs.

We performed accumulation experiments to examine if a secreted factor was responsible for inhibiting P-gp activity. Exposure of BECs to GBS-conditioned medium had no influence on P-gp function, indicating a requirement for GBS-BEC interactions (Fig. 2b). Next, we investigated if bacterial components, but not living bacteria, could generate P-gp inhibition. Interaction of heat-treated, nonviable GBS or paraformaldehyde-fixed GBS with BECs also did not affect P-gp function (Fig. 2b). These results suggest that interaction with live GBS is required for P-gp inhibition as secreted GBS components or nonviable forms of GBS were insufficient to decrease P-gp function. Given this requirement, and the fact that previous work has identified various GBS virulence factors that contribute to overall bacterial-BEC interactions, we next tested a number of bacterial mutants identified to contribute to bacterial interaction with BECs. We examined GBS mutants lacking surface expressed adhesins SfbA and Srr2, and an invasion associated gene (iagA) that functions to properly anchor bacterial lipoteichoic acid, to see if any of the virulence factors contributed to P-gp inhibition during infection [31‐34]. BECs infected by each of the mutants exhibit similar P-gp substrate accumulation to BECs infected by the wild-type (WT) GBS, and all show significantly increased accumulation over uninfected controls (Fig. 2c). Taken together, these data suggest that live GBS is required for P-gp inhibition, and SfbA, Srr2 or iagA alone does not mediate that P-gp inhibition.

To examine whether the decrease in P-gp activity correlated with a decrease in P-gp abundance, western blot and flow cytometry were used to quantify P-gp expression. BECs were infected with GBS, and western blot analysis was conducted on BEC lysates. We observed that overall P-gp abundance decreased during GBS infection (Fig. 3a, b). These results were confirmed by flow cytometry for P-gp where a decrease in P-gp expression was measured (Fig. 3c, d). RT-qPCR conducted on lysates further demonstrated that P-gp-encoding ABCB1 expression was decreased upon GBS infection (Fig. 3e). To determine if this decrease was observed in vivo, we employed our murine model of GBS meningitis [19, 33, 35]. We observed that during GBS infection mice exhibited less P-gp immunolabeling that co-localized with endothelial cells (Fig. 3f, g, Additional file 2: Figure S2). Taken together, these data suggest that the disruption in P-gp activity in BECs after GBS infection may be due to a decrease in P-gp abundance.

Induced pluripotent stem cell (iPSC) line IMR-90-C4 (WiCell) was maintained per previous reports and grown on Matrigel (WiCell) coated plates (Corning) in mTeSR1 medium (WiCell) changed daily. IMR-90 iPSCs were passaged twice a week as needed [21‐23, 26, 36, 51]. MDCK-MDR1 [52] (ATCC) cells were maintained on tissue culture treated 25 cm² flasks in DMEM (Life Technologies) + 10% Fetal Bovine Serum (WiCell). Group B Streptococcus (GBS, Streptococcus agalactiae) hypervirulent clinical isolate COH1 (serotype III, multilocus sequence type 17) strain was used [53]. Previously described COH1 mutants Δiag [33], Δsrr2 [54, 55], and ΔsfbA [34] were employed for mutant analysis. All GBS strains were grown in static Todd-Hewitt broth (THB) at 37 °C. Lactococcus lactis was grown in M17 medium at 30 °C in static culture as previously described [56]. L. lactis was prepared exactly like GBS as mentioned above.

iPSC-derived brain microvascular endothelial cells (BECs) were differentiated as previously described [21‐23, 26, 36, 51]. To ensure the quality of the cultures, mycoplasma testing was conducted periodically through services from WiCell, and PCR based kit (PanReac AppliChem). Briefly, single cell suspension of iPSCs were seeded at a density of 10,000/cm² onto Matrigel (WiCell) cell culture plates or flasks (Corning) and expanded for 3 days in mTeSR1 changing media daily. Initiation of differentiation was conducted by exchanging to unconditioned media (UM; DMEM-F12 base medium [Life Technologies], 20% Knockout serum replacement [Life Technologies], 1% minimal essential medium-nonessential amino acids [Life Technologies], 0.5% Glutamax [Life Technologies], and 0.07% beta-mercaptoethanol [Sigma]), for 6 days changing media daily. After the 6 days, media was then changed to EC medium (human endothelial cell serum-free media [Life Technologies], 1% platelet-poor plasma derived serum [Fisher], 500 ng/ml basic fibroblast growth factor, and 10 μM all trans-retinoic acid [Sigma]) for 2 days. Finally, differentiated BECs were purified onto collagen IV (Sigma) and Fibronectin (Sigma) coated plates and transwells inserts (Corning), and the following day, media was changed to EC medium without basic fibroblast growth factor or retinoic acid. BECs were analyzed for TEER using an EVOM2 instrument (World Precision) to ensure high electrical resistance unique to BECs.

iPSC-derived BECs that are purified onto collagen-fibronectin 24 well plates at 500 k cells/well were grown until Day 10 of the differentiation [23]. A multiplicity of infection (MOI) of 10 was used for all experiments unless specifically noted otherwise as previously described [36]. Overnight cultures of GBS were grown in THB media at 37 °C + 5% CO₂. The following day bacteria are subcultured and grown to an optical density at 600 nm (OD₆₀₀) of 0.400–0.600. Bacteria are then spun down and washed in phosphate-buffered saline (PBS) prior to infecting BECs. BECs were infected for 5 h at 37 °C at 5% CO₂ followed by sample collection or assays. Heat killed and formalin-fixed GBS were prepared by growing WT GBS to an OD₆₀₀ of 0.400–0.600 in THB followed by centrifugation and resuspension of the pellet in PBS and treated by heating to 95 °C for 15 min, or treatment of GBS with 4% paraformaldehyde for 15 min. Bacteria were then washed and used to treat BECs at an estimated MOI of 10 for 5 h like live infections described above. Killed GBS preparations were confirmed by plating undiluted fractions onto THB plates where no growth was observed overnight at 37 °C + 5% CO₂. GBS conditioned media was generated by growing WT GBS in endothelial cell assay media for 5 h, then sterile filtered through a 0.2 μM filter as previously described [19]. Conditioned media preparations were also confirmed sterile by plating undiluted fractions onto THB plates where no growth was observed overnight at 37 °C + 5% CO₂.

P-gp activity is assessed by the accumulation of the fluorescent substrates Rhodamine 123 (R123) (Sigma) and FLUO-3-AM (Thermo) [23, 57]. The specific P-gp inhibitor cyclosporine A (CsA) is used as a control to monitor P-gp activity [23]. For other inhibitor experiments PSC-833 (Sigma), MK571 (Sigma), or Ko143 (Sigma) were used to inhibit P-gp, MRPs, and BCRP respectively. Uninfected BECs are used as a control for comparison. After infection, cells are washed with Hank’s Balanced Salt Solution (HBSS) (Thermo) and pre-incubated with or without inhibitor (10 μM CsA, 10 μM PSC-833, 10 μM MK571, or 1 μM Ko143) for 1 h at 37 °C + 5% CO₂. Following pre-incubation, cells were incubated with 10 μM R123 or FLUO-3-AM with or without inhibitors mentioned for 2 h at 37 °C + 5% CO₂. After incubation, cells are washed twice in PBS and 200 μl of RIPA buffer (Thermo) is added and placed on a shaker for 10 min at room temperature protected from light. Fluorescence was measured on a plate reader (Tecan). Next, a BCA protein assay (Thermo) was conducted on each well and fluorescence values are normalized to BCA to account for relative cell number as previously described [23].

To assay for the expression of P-gp, BECs were either infected with GBS or left uninfected as a control. After the infection, cells are washed 3× in PBS and 100 μl of Accutase to each well and incubate at 37 °C + 5% CO₂ to remove cells from the plate. Cells are then fixed by adding 900 μl of 1% paraformaldehyde in PBS for 15 min at room temperature. Cells are then washed twice in wash buffer (5% bovine serum albumin (BSA), 0.1% Triton-X in PBS) to block and permeabilize for the staining of total P-gp. Samples were stained in the wash buffer with anti-P-gp clone F4 (Thermo) (1 μg/million cells) overnight at 4 °C. Following primary stain, samples were washed and stained with secondary anti-mouse 488 (Thermo) at 1:5000 in wash buffer for 1 h at room temperature. Cells were washed and data was collected on a MACSQuant Analyzer 10 (Miltenyi Biotec) and analyzed on FlowJo v10.

BECs were infected as described above for 5 h at an MOI of 10. After infection, cells were washed three times in PBS and lysates were taken using RIPA buffer (Thermo) plus HALT protease inhibitor cocktail (Thermo). Proteins were quantified using a standard BCA assay kit (Thermo), and equal amounts of protein were loaded onto Nu-PAGE gels (Thermo) and transferred onto nitrocellulose membranes. Membranes were blocked in 5% milk in tris-buffered saline with 0.1% Tween 20 (TBST) and anti-COX-IV antibody 1:1000 (Cell Signaling Technologies) was used as a protein loading control, and anti-P-gp (F4) (Thermo Fisher) was used to determine P-gp abundance. To visualize protein abundance horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson Laboratory) and a BioRad ChemiDoc XRS + instrument were used to image blots.

Monolayers of BECs were either left uninfected, or infected with GBS for 5 h at an MOI of 10. After infection, cell lysates and RNA was purified using a NucleoSpin RNA kit (Machery Nagel). cDNA was generated using VILO first-strand synthesis kit (Thermo Fisher), or LunaScript RT (New England BioLabs). SYBR green qPCR was conducted for human ABCB1 (P-gp, MDR1) forward primer 5′-GAAGAGATTGTGAGGGCAGC-3′, and reverse primer 5′-CCACCAGAGAGCTGAGTTCC-3′. 18S rRNA was used to normalize results and primers used, forward primer 5′-GTAACCCGTTGAACCCCATT-3′ and reverse primer 5′-CCATCCAATCGGTAGTAGCG-3′ were previously described [58]. qPCR data was collected on an Applied Biosystems StepOnePlus and data are presented as fold change using the cycle threshold (ΔΔC_t) calculation.

Animal experiments were approved by the committee on the use and care of animals at the University of Colorado School of Medicine (protocol #00316) and performed using accepted veterinary standards. The University of Colorado School of Medicine is AAALAC accredited and the facilities meet and adhere to the standards in the “Guide for the Care and Use of Laboratory Animals”. We utilized a mouse GBS infection model as previously described [19, 33, 35]. Briefly, 8 week old male CD-1 mice (Charles River) were injected intravenously with 10⁸–10⁹ of GBS. At the experimental endpoint mice were euthanized and brain tissue was collected. One half of the brain was frozen in OCT compound (Sakura) and sectioned using a CM1950 cryostat (Leica). Sections were fixed with ice cold methanol (Sigma) for 20 min, blocked with Mouse on Mouse blocking reagent (Vector Labs), and incubated with mouse anti P-glycoprotein antibody clone C219 (ThermoFisher) overnight at 4 °C followed by goat anti-mouse conjugated to Cy3 (Jackson Immunoresearch) and tomato lectin conjugated to DyLight488 (Vector Labs). Coverslips were mounted with Fluoroshield + DAPI (Abcam) and images were taken using a BZ-X710 microscope (Keyence). Pixel intensities of P-gp staining were estimated by using the FIJI image analysis program. Briefly, lectin positive vessels were traced manually and the selection was transposed onto the P-gp image and mean pixel intensity was measured.

iPSC-BECs were fixed and stained for markers exactly as described previously [23], with the exceptions that anti-CD-31 (Abcam, cat# ab32457), and anti-ZO1 (Proteintech, cat# 21773-1-AP) were utilized in place of referenced reagents. Briefly, BECs were fixed for 15 min in cold methanol and stained overnight at 4 °C. The following day, secondary antibodies anti-mouse 488 (Invitrogen, cat# A11001), and anti-rabbit 555 (Invitrogen, cat# A31572) were used at a dilution of 1:200. Samples were then visualized on a Nikon Eclipse Ti using Nikon NIS image acquisition software.

GraphPad Prism version 5.0 (GraphPad Software Inc.) was used for all statistical analysis. For pairwise comparison, 2-tailed Student’s t test was used where appropriate. For multiple comparisons, analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test was used where appropriate. Data are represented as mean, ± standard deviation (SD) since all raw values are presented. Statistical significance was accepted at a P value of less than 0.05.