Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 69 (2), 845-52

Pneumolysin Is the Main Inducer of Cytotoxicity to Brain Microvascular Endothelial Cells Caused by Streptococcus Pneumoniae

Affiliations

Pneumolysin Is the Main Inducer of Cytotoxicity to Brain Microvascular Endothelial Cells Caused by Streptococcus Pneumoniae

G Zysk et al. Infect Immun.

Abstract

In pneumococcal meningitis it is assumed that bacteria cross the blood-brain barrier (BBB), which consists mainly of cerebral endothelial cells. The effect of Streptococcus pneumoniae on the BBB was investigated with an in vitro BBB model using a human brain microvascular endothelial cell line (HBMEC) and primary cultures of bovine brain microvascular endothelial cells (BBMEC). Within a few hours of incubation with pneumococci, rounding and detachment of the HBMEC were observed, and the transendothelial electrical resistance of the BBMEC monolayer decreased markedly. An S. pneumoniae mutant deficient in pneumolysin did not affect the integrity of the endothelial cell monolayer. Neither cell wall fragments nor isolated pneumococcal cell walls induced changes of endothelial cell morphology. However, purified pneumolysin caused endothelial cell damage comparable to that caused by the viable pneumococci. The cell detachment was dependent on de novo protein synthesis and required the activities of caspase and tyrosine kinases. The results show that pneumolysin is an important component for damaging the BBB and may contribute to the entry of pneumococci into the cerebral compartment and to the development of brain edema in pneumococcal meningitis.

Figures

FIG. 1
FIG. 1
Detachment of HBMEC after addition of pneumococci. (A) Significant HBMEC detachment was measured 6 h after addition of 107 CFU of D39/ml; the detachment progressed over 24 h. (B) After 24 h of incubation time, significant cell detachment was observed after addition of pneumococci at concentrations of 106 to 107 CFU/ml. Values are means ± standard deviations of duplicate results. ∗, ∗∗, and ∗∗∗, P values of <0.05, 0.01, and 0.001 with respect to the controls.
FIG. 2
FIG. 2
TEER of BBMEC cultures after addition of D39 (107 CFU/ml). (A) The TEER decreased rapidly after the wild-type D39 was added, both without (■) and with (⧫) antibiotic supplementation. (B) Heat-killed wild-type D39 (▾) or viable pneumolysin-deficient pneumococci (D39ply::pJDC9) (▴) caused only minor changes to the tightness of the endothelial cell monolayer. The experiments were conducted in parallel using the same preparation of BBMEC. Values are means ± standard deviations of triplicate results in relation to the mean of results for four unstimulated controls.
FIG. 3
FIG. 3
Effect of antibiotical treatment, heat inactivation, and pneumolysin activity on HBMEC detachment. Endothelial cell detachment was quantitated 24 h after addition of D39 and the pneumolysin-deficient mutant (D39ply::pJDC9; 107 CFU/ml). Viable pneumococci were added without and with (#) antibiotic supplementation. Antibiotical inactivation of the wild-type D39 caused significant cell cytotoxicity, whereas antibiotical treatment of the pneumolysin-deficient mutant did not. Heat inactivation of the wild-type D39 (D39 hi) attenuated the cytotoxic potential distinctly. Values are means ± standard deviations of duplicate results. ∗, P value of <0.001 with respect to results for viable D39.
FIG. 4
FIG. 4
Effect of pneumolysin-neutralizing antiserum on HBMEC detachment 24 h after addition of wild-type D39 (107 CFU/ml). The antiserum abolished the pneumococcal cytotoxicity completely at a final dilution of 1:10; at dilutions of more than 1:30, no effect was evident over 24 h. Values are means ± standard deviations of duplicates. ∗, P value of <0.001 with respect to results for viable D39 in the absence of antiserum.
FIG. 5
FIG. 5
Effect of purified pneumolysin on HBMEC detachment 24 h after addition of the protein in comparison to results for pneumococci (D39; 107 CFU/ml). Values are means of duplicate results; error bars are standard deviations. ∗ and ∗∗, P values of <0.01 and <0.001 with respect to controls. The hemolytic titers of the culture medium after supplementation with 30 μg of pneumolysin/ml and 107 CFU of pneumococci/ml were comparable.
FIG. 6
FIG. 6
Inhibition of HBMEC protein synthesis, tyrosine phosphorylation, and caspase activity. Cycloheximide (C), herbimycin A (HA), and z-VAD-fmk (z-VAD) were used at 5 μg/ml, 2.5 μg/ml, and 10 μM, respectively. HBMEC were preincubated with herbimycin A for 4 h and with z-VAD-fmk for 2 h prior to addition of 107 CFU of D39/ml. Detachment of HBMEC was quantified 8 h thereafter. Values are means ± standard deviations of duplicates. ∗, P value of <0.001 with respect to viable D39 in the absence of inhibitors.

Similar articles

See all similar articles

Cited by 43 PubMed Central articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback