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Penetration of the blood–brain barrier by Staphylococcus aureus: contribution of membrane-anchored lipoteichoic acid

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Abstract

Staphylococcus aureus is one of the most prevalent organisms responsible for nosocomial infections, and cases of community-acquired S. aureus infection have continued to increase despite widespread preventative measures. Pathologies attributed to S. aureus infection are diverse; ranging from dermal lesions to bacteremia, abscesses, and endocarditis. Reported cases of S. aureus-associated meningitis and brain abscesses have also increased in recent years, however, the precise mechanism whereby S. aureus leave the bloodstream and gain access to the central nervous system (CNS) are not known. Here we demonstrate for the first time that S. aureus efficiently adheres to and invades human brain microvascular endothelial cells (hBMEC), the single-cell layer which constitutes the blood–brain barrier (BBB). The addition of cytochalasin D, an actin microfilament aggregation inhibitor, strongly reduced bacterial invasion, suggesting an active hBMEC process is required for efficient staphylococcal uptake. Furthermore, mice injected with S. aureus exhibited significant levels of brain bacterial counts and histopathologic evidence of meningeal inflammation and brain abscess formation, indicating that S. aureus was able to breech the BBB in an experimental model of hematogenous meningitis. We found that a YpfP-deficient mutant, defective in lipoteichoic acid (LTA) membrane anchoring, exhibited a decreased ability to invade hBMEC and correlated to a reduced risk for the development of meningitis in vivo. Our results demonstrate that LTA-mediated penetration of the BBB may be a primary step in the pathogenesis of staphylococcal CNS disease.

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Acknowledgments

We are grateful to Monique Stins and Kwang Sik Kim for providing hBMEC and Satoshi Uchiyama for the SPN D39 glycosyltransferase ΔGTG mutant. The histopathologic analysis was performed at the University of California San Diego Histopathology Core Facility, Nissi Varki, director. This work was supported by grant no. R01 NS051247 from the National Institutes of Health to K.S.D. The authors declare that they have no conflict of interest.

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Author notes

  1. Ida H. Hiemstra

    Present address: Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands

Authors and Affiliations

  1. Department of Biology and Center for Microbial Sciences, San Diego State University, 5500 Campanile Drive, San Diego, CA, 92182, USA

    Tamsin R. Sheen, Celia M. Ebrahimi, Ida H. Hiemstra, Steven B. Barlow & Kelly S. Doran

  2. Cellular and Molecular Microbiology Biology Division, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, 72076, Tübingen, Germany

    Andreas Peschel

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  1. Tamsin R. Sheen
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Correspondence to Kelly S. Doran.

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Supplemental Figure 1

Role of YpfP in S. aureus HUVEC attachment and invasion. Adherence (a) and invasion (b) of human umbilical vein endothelial cells by S. aureus strain SA113 and isogenic mutant strain ΔypfP. Data are expressed as the total cell-associated (adherent) or intracellular (invasive) CFU recovered compared to that obtained with the WT (parental) strain. HUVEC monolayers were incubated with bacterial strains (MOI = 0.1) for 60 min followed by a 60-min gentamicin treatment to recover intracellular CFU (% invasion). The ΔypfP did not exhibit a decreased ability to adhere to HUVEC, but was significant less invasive. All assays were repeated in triplicate at least three times; data from a representative experiment are shown, *p < 0.05 (GIF 19 kb)

High-Resolution (TIFF 730 kb)

Supplemental Figure 2

Contribution of membrane-anchored LTA to S. aureus murine kidney invasion. Male CD-1 mice were injected i.v. with 3 × 107 cfu S. aureus SA113 or ΔypfP (a), or equal amounts of both strains (1 × 107 cfu final inoculum; b). Tissue was harvested 96 h p.i. and cfu enumerated on LB agar plates. Duplicate samples were plated on LB agar supplemented with erythromycin (15 µg mL−1) to differentiate WT and ΔypfP CFU in (b). There was no significant difference in bacterial load in the kidney between the two strains (95% confidence interval). For the competition experiment shown in (b), an overall ratio of 1 indicates equal numbers of WT and ΔypfP CFU were recovered from the tissue. (GIFF 18 kb)

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Sheen, T.R., Ebrahimi, C.M., Hiemstra, I.H. et al. Penetration of the blood–brain barrier by Staphylococcus aureus: contribution of membrane-anchored lipoteichoic acid. J Mol Med 88, 633–639 (2010). https://doi.org/10.1007/s00109-010-0630-5

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