Host Cytosolic Phospholipase A2α Contributes to Group B Streptococcus Penetration of the Blood-Brain Barrier

doi:10.1128/IAI.05506-11

Infect Immun. 2011 October; 79(10): 4088–4093.

PMCID: PMC3187255

Host Cytosolic Phospholipase A₂α Contributes to Group B Streptococcus Penetration of the Blood-Brain Barrier

Ravi Maruvada,¹ Longkun Zhu,¹ Donna Pearce,¹ Adam Sapirstein,² and Kwang Sik Kim¹^*

¹Department of Pediatrics, Division of Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, 200 North Wolfe Street, Room 3157, Baltimore, Maryland 21287

²Department of Anesthesiology and Critical Care Medicine, 600 North Wolfe Street, Meyer 297-A, Baltimore, Maryland 21287

F. C. Fang, Editor

^*Corresponding author. Mailing address: Department of Pediatrics, Division of Pediatric Infectious Diseases, Room 3157, Johns Hopkins University School of Medicine, 200 North Wolfe St., Baltimore, MD 21287. Phone: (410) 614-3917. Fax: (410) 614-1491. E-mail: kwangkim/at/jhmi.edu.

Received June 13, 2011; Revisions requested July 20, 2011; Accepted July 27, 2011.

This article has been cited by other articles in PMC.

Abstract

Group B Streptococcus (GBS) is the most common bacterium causing neonatal meningitis, and neonatal GBS meningitis continues to be an important cause of mortality and morbidity. Here we provide the first direct evidence that host cytosolic phospholipase A₂α (cPLA₂α) contributes to type III GBS invasion of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier and penetration into the brain, the key step required for the development of GBS meningitis. This was shown by our demonstration that pharmacological inhibition and gene deletion of cPLA₂α significantly decreased GBS invasion of the HBMEC monolayer and penetration into the brain. cPLA₂α releases arachidonic acid from membrane phospholipids, and we showed that the contribution of cPLA₂α to GBS invasion of HBMEC involved lipoxygenated metabolites of arachidonic acid, cysteinyl leukotrienes (LTs). In addition, type III GBS invasion of the HBMEC monolayer involves protein kinase Cα (PKCα), as shown by time-dependent PKCα activation in response to GBS as well as decreased GBS invasion in HBMEC expressing dominant-negative PKCα. PKCα activation in response to GBS, however, was abolished by inhibition of cPLA₂α and cysteinyl LTs, suggesting that cPLA₂α and cysteinyl LTs contribute to type III GBS invasion of the HBMEC monolayer via PKCα. These findings demonstrate that specific host factors involving cPLA₂α and cysteinyl LTs contribute to type III GBS penetration of the blood-brain barrier and their contribution involves PKCα.

INTRODUCTION

Neonatal bacterial meningitis continues to be an important cause of mortality and morbidity. Group B Streptococcus (GBS) is the most common bacterium causing neonatal meningitis (3, 5, 6, 12, 13, 18–20, 22, 25, 26, 37), and inadequate knowledge of its pathogenesis has contributed to this mortality and morbidity (20, 26). Experimental data indicate limited efficacy with antimicrobial chemotherapy alone (19).

Several lines of evidence from human cases of GBS meningitis as well as animal models of experimental hematogenous GBS meningitis indicate that GBS penetrates into the brain initially in the cerebral vasculature (2, 7, 8).

We have developed an in vitro model of the blood-brain barrier using human brain microvascular endothelial cells (HBMEC) (21, 34, 38). These HBMEC have been shown to exhibit morphological and functional properties of tight junction formation as well as a polarized monolayer (21, 34, 38). In addition, we and others have developed animal models of experimental hematogenous GBS meningitis (7, 8, 19). These animal models have important similarities to GBS meningitis in human neonates, such as hematogenous infection of the meninges. Using these in vitro and in vivo models, we have shown that type III GBS strains, which account for the majority of cerebrospinal fluid (CSF) isolates from neonates with GBS meningitis, invade the HBMEC monolayer and penetrate the brain (19, 31, 35, 36, 40). Type III GBS internalization into the HBMEC monolayer has also been documented by transmission electron microscopy (31). The mechanisms involved in GBS invasion of the HBMEC monolayer and penetration into the brain, however, remain incompletely understood.

In the present study, using pharmacological inhibition and gene deletion, we showed that host cytosolic phospholipase A₂α (cPLA₂α) contributes to type III GBS invasion of HBMEC and penetration into the brain, likely involving lipoxygenated metabolites of arachidonic acid released by cPLA₂α as well as protein kinase Cα (PKCα).

MATERIALS AND METHODS

Reagents.

MK571 and montelukast were purchased from Cayman Chemical Company (Ann Arbor, MI), and arachidonyltrifluoromethyl ketone (AACOCF3) was purchased from Biomol Laboratories (Plymouth Meeting, PA). CP105696 was a gift from Pfizer (Fig. 1). cPLA₂α, phospho-cPLA₂α, and phospho-PKCα (p-PKCα) antibodies were purchased from Cell Signaling Technologies (Danvers, MA), and PKC antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Fig. 1.

Arachidonic acid metabolism pathways were examined for their contributions to type III GBS invasion of the HBMEC monolayer and/or penetration into the brain. The molecules examined include cytosolic phospholipase A₂α (cPLA₂α) and the leukotrienes (more ...)

Isolation and culture of HBMEC.

HBMEC were isolated and characterized as described previously (38). Briefly, brain specimens were cut into small pieces and homogenized in Dulbecco minimal essential medium (DMEM) containing 2% fetal bovine serum (FBS) (DMEM-S) using a Dounce homogenizer. The homogenate was centrifuged in 15% dextran in DMEM-S for 10 min at 10,000 × g. The pellet containing crude microvessels was further digested in a solution containing 1 mg/ml collagenase-dispase in DMEM-S for 1 h at 37°C. Microvascular capillaries were isolated by adsorption to a column of glass beads (0.25 to 0.3 mm) and washing off the beads. HBMEC were plated on rat tail collagen-fibronectin-coated dishes or glass coverslips and cultured in RPMI 1640-based medium with growth factors, 10% heat-inactivated FBS, 10% NuSerum, 5 U/ml heparin, 2 mM l-glutamine, 1 mM sodium pyruvate, nonessential amino acids, vitamins, and 100 U/ml penicillin and streptomycin. HBMEC were positive for factor VIII-Rag, took up fluorescently labeled acetylated low-density lipoprotein, and expressed γ-glutamyl transpeptidase. HBMEC were maintained in RPMI 1640-based medium, including 10% FBS and 10% NuSerum (BD Biosciences), at 37°C in a humid atmosphere of 5% CO₂ as described previously (38).

GBS adherence and invasion assays in HBMEC.

HBMEC (~70% confluence) were infected with dialyzed adenovirus constructs containing cPLA₂α-S505A or green fluorescent protein (GFP) vector, or dominant-negative PKCα or a vector control, as previously described (35, 36, 41). Clinical isolates of type III GBS strains were used for HBMEC adherence and invasion assays. Strains K79, K160, K231, and K243 were the CSF isolates from neonates with meningitis (17, 19, 31) and belong to the hypervirulent clone sequence type 17 (ST-17) (23, 29), while strain ATCC 12403 (or NEM 316), an isolate from a case of fatal septicemia, belongs to ST-23 (10, 39). Streptococcus gordonii was used as a noninvasive control (31). Bacterial strains were grown overnight in Todd-Hewitt (TH) broth (Difco Laboratories, Detroit, MI), resuspended in experimental medium (M199-Ham F-12 [1:1] containing 5% heat-inactivated FBS, 2 mM glutamine, and 1 mM pyruvate), and added at a multiplicity of infection (MOI) of 5 to 10 to HBMEC grown in collagen-coated 24-well plates at 37°C in a 5% CO₂ incubator for 2 h for an adherence assay, as previously described (31, 35, 36, 40). HBMEC were washed four times with phosphate-buffered saline (PBS) to remove unbound bacteria, lysed in 0.025% Triton X-100, and cultured for determinations of CFU. The results were calculated as the percent adherence [(number of bacteria recovered/number of bacteria inoculated) × 100] and expressed as the relative adherence (adherence as a percentage of that of the GBS strain in HBMEC transfected with a vector control or in the presence of a vehicle control). Each set was run in triplicate.

The HBMEC invasion assay with penicillin and gentamicin treatment (31, 35, 36, 40) was performed to determine the number of viable intracellular bacteria recovered from the infected HBMEC. GBS strains were added to HBMEC as described above for the adherence assay. HBMEC were subsequently washed with RPMI 1640 and incubated with experimental medium containing penicillin (5 μg/ml) and gentamicin (100 μg/ml) for 1 h to kill extracellular bacteria. The cells were washed again with PBS and lysed in 0.025% Triton X-100, and the released intracellular bacteria were enumerated by culturing them on sheep blood agar plates. The results were calculated as the percent invasion [(number of intracellular bacteria recovered/number of bacteria inoculated) × 100] and expressed as the relative invasion (invasion as a percentage of that of the GBS strain in HBMEC transfected with a vector control or in the presence of a vehicle control). Each set was run in triplicate.

Immunoblotting and immunoprecipitation.

The lysates of HBMEC incubated with GBS or S. gordonii were prepared for Western blotting and immunoprecipitation, as described previously (35, 36, 41).

Mouse model of experimental hematogenous meningitis.

cPLA₂α^−/− and wild-type 10- to 13-week-old mice, either male or female, that had been backcrossed on the BALB/c strain for >10 generations were used (41). All procedures and handling techniques were approved by the Johns Hopkins University Animal Care and Use Committee. Each mouse received 1 × 10⁷ CFU of GBS strain K79 in 100 μl PBS via the tail vein. One hour later, the mouse chest was cut open, and blood from the right ventricle was collected for determination of bacterial counts, which were expressed as CFU/ml of blood. The mouse was then perfused with mammalian Ringer's solution by transcardiac perfusion through a 23-gauge needle inserted into the left ventricle of the heart under a perfusion pressure of about 100 mm Hg as described previously (41). The perfusate exited through a cut in the right atrium. The brains were removed, weighed, homogenized in 2 ml RPMI 1640, and cultured for determination of bacterial counts, which were expressed as CFU/g. Since GBS penetration into the brain is shown to depend on the magnitude of bacteremia (8, 19), bacterial penetration into the brain was also expressed as (brain CFU per g/blood CFU per ml) × 100. Kidneys and spleens were also removed, homogenized, and cultured for determination of bacterial counts, which were expressed as CFU/g.

Statistical analysis.

Data were expressed as means ± standard errors of the means (SEM). Differences of bacterial counts in the blood, brain, kidney, and spleen and the percentage of bacterial penetration between different experimental groups were determined by the Wilcoxon rank sum test. Differences in HBMEC adherence and invasion between bacterial strains with and without inhibitors or between HBMEC transfected with different constructs were determined by one-way analysis of variance (ANOVA) followed by Dunnett's test. The level of significance was set at a P value of <0.05.

RESULTS

Role of host cPLA₂α in type III GBS invasion of HBMEC.

We showed that host cPLA₂α contributes to HBMEC invasion as well as penetration into the brain by neonatal meningitis-causing Escherichia coli K1 (4, 41). Since GBS and E. coli represent the two most common bacteria causing neonatal meningitis (3, 5, 6, 12, 13, 18–20, 22, 25, 26, 37), we examined whether host cPLA₂α contributes to GBS invasion of the blood-brain barrier. The role of cPLA₂α in type III GBS adherence to and invasion of HBMEC was examined by using an inhibitor of cPLA₂α (arachidonyl trifluoromethylketone [AACOCF3]), as previously described (31, 35, 36). Briefly, HBMEC pretreated with different concentrations of AACOCF3 (10 to 40 μM) or a vehicle control (dimethyl sulfoxide [DMSO]) were incubated with the type III GBS strain ATCC 12403 (which belongs to ST-23 and whose genome is sequenced [10, 39]) and examined for bacterial adherence and invasion. While AACOCF3 did not affect the adherence of GBS to HBMEC, GBS invasion was inhibited in a dose-dependent manner, with a decrease of approximately 70% in HBMEC pretreated with 40 μM AACOCF3 compared to that in vehicle-treated cells (Fig. 2A).

Fig. 2.

Host cPLA₂α contributes to type III GBS invasion of HBMEC. (A) HBMEC pretreated with a vehicle control or various concentrations of AACOCF3 were incubated with type III GBS strain ATCC 12403. For invasion assays, numbers of viable intracellular (more ...)

We next examined whether AACOCF3 affects CSF isolates of GBS for their invasion of HBMEC. For these studies, we used type III GBS isolates belonging to the hypervirulent ST-17 clone (19, 23). Type III GBS invasion frequency (mean) ranged between 1 and 7% of the inocula in HBMEC. AACOCF3 at 40 μM inhibited HBMEC invasion by all CSF isolates of the ST-17 clone, while HBMEC invasion of a noninvasive S. gordonii control strain was not affected by AACOCF3 (Fig. 2B). It is important to note that AACOCF3 at 40 μM did not affect the integrity of the HBMEC monolayer, as shown by Live/Dead staining (Molecular Probes), and also did not affect the bacterial growth, as determined by comparing numbers of CFU in experimental medium with or without AACOCF3. These findings suggest that cPLA₂α is likely to play a role in HBMEC invasion by type III GBS strains of both ST-17 and non-ST-17 clones.

Examination of serine phosphorylation of cPLA₂α in response to type III GBS invasion of HBMEC.

cPLA₂α activation in response to E. coli invasion of HBMEC has been shown to involve serine phosphorylation at S505 (41). Hence, to examine the role of S505 in GBS invasion, HBMEC were transfected with adenovirus constructs containing the cPLA₂α mutant (32), in which serine 505 was replaced with alanine (S505A) or a GFP control, and used for invasion experiments with type III GBS strains ATCC 12403 and K79. The results showed that type III GBS invasion was significantly decreased in HBMEC transfected with the S505A construct compared to that in HBMEC transfected with a GFP control (Fig. 3A). These findings demonstrate that cPLA₂α phosphorylation at the serine 505 site is likely to be involved in type III GBS invasion of HBMEC.

Fig. 3.

Role of cPLA₂α serine phosphorylation in type III GBS invasion of HBMEC. (A) To examine the effect of cPLA₂α phosphorylation at serine 505 in GBS invasion, HBMEC (~70% confluence) were transfected with dialyzed adenovirus constructs (more ...)

Serine 505 phosphorylation of cPLA₂α in response to type III GBS was further examined by Western blotting. Briefly, HBMEC were incubated with GBS strains ATCC 12403 or K79 for various time points. Cell lysates were then examined for cPLA₂α phosphorylation using a specific cPLA₂α antibody directed against serine 505 phosphorylation. In addition, the noninvasive S. gordonii strain was included as a negative control. Densitometric analysis of bands obtained in Western blots showed that the levels of S505 phosphorylation were increased ~4-fold in a time-dependent manner in response to type III GBS strains ATCC 12403 and K79, while S505 phosphorylation was not evident in HBMEC incubated with S. gordonii (Fig. 3Bi and ii).

Taken together, these findings demonstrate that host cPLA₂α phosphorylation at serine 505 occurs in response to type III GBS strains in HBMEC and cPLA₂α activation plays a role in type III GBS invasion of HBMEC.

Role of host cPLA₂α in type III GBS penetration into the brain.

Since AACOCF3 (a cPLA₂α inhibitor) inhibited type III GBS invasion of HBMEC, we next examined the role of cPLA₂α in type III GBS penetration into the brain in the mouse model of experimental hematogenous meningitis using cPLA₂α knockout mice compared to strain-matched wild-type mice. Briefly, each animal received 1 × 10⁷ CFU of type III GBS strain K79 via the tail vein. One hour later, blood was obtained to determine the number of CFU. The animals were then perfused with sterile Ringer's solution until the perfused solution became colorless. Next, brains, kidneys, and spleens were removed, weighed, homogenized, and cultured for determinations of CFU.

The magnitude of bacteremia did not differ significantly between cPLA₂α^−/− and wild-type animals, as shown by similar bacterial counts in the blood (CFU/ml) (Fig. 4A). However, bacterial counts in the brain were significantly less in cPLA₂α^−/− mice than in wild-type animals (Fig. 4B). These findings indicate that decreased GBS penetration into the brain of cPLA₂α^−/− mice was not the result of lower levels of bacteremia in cPLA₂α^−/− mice than in wild-type mice. This concept was also demonstrated when the results were expressed as a percentage of bacterial counts in the brain compared to those in the blood, which was significantly less in cPLA₂α^−/− mice than in wild-type animals (Fig. 4C). Of interest, the bacterial counts in the kidneys and spleens (CFU/g) did not differ between the two groups of animals (Fig. 4D and E).

Fig. 4.

Decreased penetration of type III GBS strain K79 into the brains of cPLA₂α^−/− mice. GBS counts in the blood (A), brain (B), kidney (D), and spleen (E) as well as the percentage of bacterial penetration (brain counts [CFU/g]/blood (more ...)

Taken together, these in vitro and in vivo findings demonstrate that host cPLA₂α contributes to type III GBS invasion of the HBMEC monolayer and penetration specifically into the brain.

Role of cysteinyl leukotrienes in type III GBS invasion of HBMEC.

Since cPLA₂α selectively liberates arachidonic acid from the sn-2 position of membrane phospholipids (9, 33), we hypothesized that the contribution of cPLA₂α to type III GBS invasion of HBMEC is likely to be related to metabolites of arachidonic acid. Leukotrienes (LTs) are synthesized from arachidonate by 5-lipoxygenase (5-LO) and 5-LO-activating protein (FLAP) (Fig. 1) (33). Leukotriene B₄ (LTB₄) and the cysteinyl LTs (LTC₄, LTD₄, and LTE₄) represent terminal LTs, and their biological actions are transduced by ligation of specific G-protein-coupled receptors (GPCRs), which include BLT-1 for LTB₄ and CysLT1 for cysteinyl LTs (33). Hence, the role of LTs in type III GBS invasion of HBMEC was examined using CysLT1 antagonists (MK571 and montelukast) and a BLT-1 antagonist (CP105696). As a negative control, the noninvasive S. gordonii strain was included.

Invasion analysis showed that while MK571 and montelukast inhibited ATCC 12403 invasion of HBMEC, CP105696 had no effect on GBS invasion (Fig. 5A, B, and C). On the other hand, S. gordonii exhibited no significant difference in invasion between MK571-treated and untreated HBMEC (Fig. 5A). The experiments were repeated with strain K79 in the presence of various concentrations of montelukast. A significant dose-dependent decrease in HBMEC invasion was observed, similar to that of strain ATCC 12403, while there were no differences in HBMEC adherence. These findings demonstrate that cysteinyl LTs, but not LTB₄, are likely to contribute to type III GBS invasion of the blood-brain barrier.

Fig. 5.

Effect of leukotriene receptor antagonists on type III GBS invasion of HBMEC. HBMEC were pretreated with a vehicle control, the CysLT1 antagonists (MK571 and montelukast), or the BLT-1 antagonist (CP105696) and then examined for adherence and invasion (more ...)

Involvement of PKCα in type III GBS invasion of HBMEC.

Our findings so far demonstrate that host cPLA₂α and cysteinyl LTs contribute to type III GBS invasion of HBMEC, but their underlying mechanisms remain unclear. PKC is a family of at least 12 serine/threonine kinases that transduce multiple signals in the regulation of a variety of cellular functions, which include actin cytoskeleton rearrangements (14, 24). We have shown that type III GBS invasion of HBMEC requires host cell actin cytoskeleton rearrangements, as shown by the demonstration that pretreatment of HBMEC with cytochalasin D (microfilament-disrupting agents) inhibited type III GBS invasion of HBMEC (31). We therefore examined whether the contributions of cPLA₂α and cysteinyl LTs to type III GBS invasion of HBMEC involve PKC, specifically PKCα.

HBMEC were transfected with adenovirus constructs expressing dominant-negative PKCα or a vector control and then examined for strain K79 invasion. Invasion analysis showed that GBS invasion was significantly decreased in HBMEC expressing dominant-negative PKCα by ~55% compared to that in vector control-transfected HBMEC, while no differences in GBS adherence to HBMEC were observed (Fig. 6A). Next, PKCα activation (i.e., serine phosphorylation) in response to GBS was examined in the presence of a vehicle control, CP105696, AACOCF3, and montelukast. Briefly, HBMEC were incubated with GBS strain K79 for various time points in the presence of a vehicle control or inhibitors. Lysates were then immunoprecipitated with PKCα antibody, and immunoprecipitates were examined for phospho-PKCα (p-PKCα). While the levels of p-PKCα were shown to increase in a time-dependent manner in HBMEC treated with a vehicle control or CP105696, such increases in p-PKCα were abolished in HBMEC that were incubated with either AACOCF3 or montelukast (Fig. 6Bi and ii).

Fig. 6.

Role of PKCα in type III GBS invasion of HBMEC. (A) To examine the role of PKCα in GBS invasion of HBMEC, the HBMEC (~70% confluence) were transfected with dialyzed adenovirus constructs expressing dominant-negative PKCα (more ...)

These findings demonstrate that host cPLA₂α and cysteinyl LTs contribute to type III GBS invasion of HBMEC, most likely involving PKCα, and also that PKCα is downstream of cPLA₂α and cysteinyl LTs in type III GBS invasion of the blood-brain barrier.

DISCUSSION

GBS is the most common bacterium causing neonatal meningitis, and neonatal GBS meningitis continues to be an important cause of mortality and morbidity. However, the microbe-host interactions involved in GBS invasion of the blood-brain barrier and penetration into the brain remain incompletely understood (18, 20, 26). Previous studies have identified several microbial factors promoting GBS invasion of HBMEC and/or penetration into the brain, including fibrinogen adhesion protein FbsA, laminin-binding protein Lmb, hemolysin-cytolysin, pilus, lipoteichoic acid-anchoring protein IagA, the serine-rich repeat 1 glycoprotein, and the hypervirulent GBS adhesin (HvgA) (26, 39, 40), but relevant host factors involved in GBS invasion of the blood-brain barrier have not been determined.

The findings reported here using pharmacological inhibition and gene deletion demonstrate for the first time that host cPLA₂α contributes to type III GBS invasion of the HBMEC monolayer and penetration into the brain, the key step required for the development of meningitis (18, 20). Deletion of a functional cPLA₂α has been shown to attenuate the development of arthritis, bone resorption, and pulmonary fibrosis (11, 15, 28, 30), but the role of host cPLA₂α in GBS penetration into the brain has not previously been reported. We showed that type III GBS penetration into the brain of cPLA₂α^−/− mice was significantly less compared to that of wild-type mice, but the magnitudes of bacteremia were similar between cPLA₂α^−/− and wild-type mice. Therefore, the significantly decreased penetration of type III GBS into the brains of cPLA₂α^−/− mice was not the result of decreased levels of bacteremia. cPLA₂α deletion, however, did not affect type III GBS penetration into the nonbrain organs, such as kidneys and spleens, as shown by similar numbers of bacterial counts recovered from cPLA₂α^−/− and wild-type mice. The basis for this selective role of host cPLA₂α in type III GBS penetration into the brain but not into nonbrain organs remains unclear.

cPLA₂α selectively liberates arachidonic acid from membrane phospholipids (9, 33), suggesting that the contribution of cPLA₂α to type III GBS invasion of HBMEC is likely to be related to metabolites of arachidonic acid. 5-LO and FLAP oxygenate arachidonic acid to LTs, and LTB₄ and cysteinyl LTs represent terminal LTs, interacting with their respective GPCRs, BLT-1 and CysLT1, respectively (Fig. 1) (33). We showed that the CysLT1 antagonists (MK571 and montelukast) inhibited type III GBS invasion of HBMEC, while the BLT-1 antagonist failed to affect type III GBS invasion of HBMEC. Taken together, these findings demonstrate that host cPLA₂α contributes to type III GBS invasion of HBMEC, most likely involving lipoxygenated metabolites of arachidonic acid, cysteinyl LTs.

Another novel finding of our study is the demonstration that the contribution of host cPLA₂α and cysteinyl LTs to type III GBS invasion of HBMEC involves PKCα. These findings differ from those of other investigators who showed that PKCα contributes to cPLA₂α phosphorylation in immortalized rat brain endothelial cells (1) but are similar to those of our earlier studies with E. coli invasion of HBMEC, where PKCα is shown to be downstream of cPLA₂α and cysteinyl LTs in E. coli K1 invasion of HBMEC (41). These findings suggest that cPLA₂α-cysteinyl LT-PKCα pathways are likely to be involved in neonatal meningitis-causing bacteria (e.g., type III GBS and E. coli K1) for their penetration of the blood-brain barrier, and additional studies are needed to elucidate how the pathways involving cPLA₂α, cysteinyl LTs, and PKCα contribute to neonatal meningitis.

Clinical isolates of type III GBS contain a limited number of clonal complexes, defined by multilocus sequence typing. ST-17 is strongly associated with neonatal meningitis and was designated the hypervirulent clone (16, 23, 27, 29). A recent study identified that an ST-17-specific surface-expressed GBS protein, HvgA, is linked to the hypervirulence of the ST-17 GBS clone in type III GBS crossing of the blood-brain barrier (39). In our study, the contribution of host cPLA₂α to GBS invasion of the blood-brain barrier was demonstrated for type III isolates of both ST-17 and ST-23 clones. It is therefore likely that the host-microbe interactions involved in host cPLA₂α-mediated invasion of the blood-brain barrier differ from those involved in HvgA-mediated GBS invasion of host cells. The determination and characterization of the GBS-host interactions involved in cPLA₂α-mediated invasion of the blood-brain barrier are therefore likely to elucidate the mechanisms for selective penetration of GBS into the brain, and studies are in progress to investigate this issue.

In summary, our findings demonstrate for the first time that type III GBS strains exploit host cPLA₂α and 5-LO-derived cysteinyl LTs for their invasion of the blood-brain barrier and penetration into the brain, and host cPLA₂α and cysteinyl LTs contribute to type III GBS invasion of the blood-brain barrier, involving PKCα.

ACKNOWLEDGMENTS

This work was supported by NIH grants NS26310 and AI84984.

The animal experiments were approved by the Animal Care and Use Committee of Johns Hopkins University.

Footnotes

Published ahead of print on 8 August 2011.

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