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. 2010 Jun;88(6):633-9.
doi: 10.1007/s00109-010-0630-5. Epub 2010 Apr 24.

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

Tamsin R Sheen  1 , Celia M Ebrahimi, Ida H Hiemstra, Steven B Barlow, Andreas Peschel, Kelly S Doran
Affiliations

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

Tamsin R Sheen et al. J Mol Med (Berl). 2010 Jun.

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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Figures

Figure 1
Figure 1. S. aureus adheres to and invades human brain microvascular endothelial cells
(A) Micrograph of Gram-stained hBMEC infected with S. aureus strain ISP479C (MOI=10). Transmission electron micrographs demonstrating hBMEC invasion by S. aureus strains ISP479C (B) and SA113 (C) (MOI=10). (D) Adherence and invasion of hBMEC by S. aureus ISP479C, SA113, ATCC 33591 and USA 300 strain TCH1516 following a 60 min incubation. Data are expressed as the total adherent or intracellular CFU recovered compared to the input inoculum (MOI=0.1). (E) S. aureus ISP479C hBMEC invasion following treatment with cytochalasin D or equivalent concentrations of DMSO. All experiments were repeated at least three times in triplicate; data from a representative experiment are shown. Error bars indicate 95% confidence intervals of mean values from three wells. ***, P <0.001.
Figure 2
Figure 2. S. aureus BBB penetration in vivo using a mouse model of hematogenous meningitis
Male CD-1 mice (n=32) were injected intravenously via tail vein with 2 × 108 cfu S. aureus ISP479C. Infection experiments were performed three times, a representative experiment is shown. Cohorts of 8 individuals were sacrificed at 24 h intervals and bacteria enumerated from (A) blood, (B) brain and (C) kidney. Histopathology of H&E (D-F) or Gram stained (F) brain tissue 96 h post-infection. Representative samples showed (D) meningeal thickening and hemorrhage and (E) abscesses formation and cellular infiltration. (F-G) close up of the abscess in (E), (G) depicts presence of staphylococci in abscess (arrow).
Figure 3
Figure 3. Contribution of membrane-anchored LTA to S. aureus BBB penetration
(A) Relative hBMEC adherence and invasion by S. aureus SA113, isogenic mutant strain ΔypfP and complemented mutant strain ΔypfP compl. (MOI=0.1). (B) Invasion of hBMEC by parental S. aureus SA113 (MOI=0.1), group B Streptococcus (GBS) (MOI=1.0) and Streptococcus pneumoniae (SPN) (MOI=10) and their respective glycosyltransferase mutants ΔypfP, Δiag and ΔGTG. All experiments were repeated at least three times in triplicate; data from a representative experiment are shown. *, p <0.05, **, p <0.01. S. aureus SA113 and ΔypfP CFU recovered from blood (C) and brain (D) tissue of CD-1 male mice 72 h post- i.v. infection (~3 × 106 cfu). (E) Analysis of bacterial counts recovered from the brain 96 h post- i.v. injection with equal amounts of WT and ΔypfP mutant S. aureus SA113 strains (1 × 107 total cfu). CFU were enumerated on LB plates or on LB supplemented with erythromycin to distinguish between WT and mutant bacteria. An overall ratio of 1 indicates equal numbers of WT and ΔypfP CFU, a ratio greater than 1 indicates a higher number of WT bacteria were recovered. (F-H). Histopathology of H&E-stained brain tissue from representative mice infected with SA113 or ΔypfP in (D). (F) Hemorrhage and meningeal thickening observed in a WT-infected mouse with a high recovered CFU. (G) Normal brain architecture was observed in a mouse infected with the ΔypfP mutant with little or no recovered CFU. (H) Meningeal thickening was evident in a ΔypfP-infected mouse with a high recovered CFU.
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