Abstract
Bacterial meningitis occurs when bloodborne pathogens invade and penetrate the blood-brain barrier (BBB), provoking inflammation and disease. Group B Streptococcus (GBS), the leading cause of neonatal meningitis, can enter human brain microvascular endothelial cells (hBMECs), but the host response to intracellular GBS has not been characterized. Here we sought to determine whether antibacterial autophagy, which involves selective recognition of intracellular organisms and their targeting to autophagosomes for degradation, is activated in BBB endothelium during bacterial infection. GBS infection resulted in increased punctate distribution of GFP-microtubule-associated protein 1 light chain 3 (LC3) and increased levels of endogenous LC3-II and p62 turnover, two hallmark indicators of active autophagic flux. Infection with GBS mutants revealed that bacterial invasion and the GBS pore-forming β-hemolysin/cytolysin (β-h/c) trigger autophagic activation. Cell-free bacterial extracts containing β-h/c activity induced LC3-II conversion, identifying this toxin as a principal provocative factor for autophagy activation. These results were confirmed in vivo using a mouse model of GBS meningitis as infection with WT GBS induced autophagy in brain tissue more frequently than a β-h/c-deficient mutant. Elimination of autophagy using Atg5-deficient fibroblasts or siRNA-mediated impairment of autophagy in hBMECs led to increased recovery of intracellular GBS. However, electron microscopy revealed that GBS was rarely found within double membrane autophagic structures even though we observed GBS-LC3 co-localization. These results suggest that although autophagy may act as a BBB cellular defense mechanism in response to invading and toxin-producing bacteria, GBS may actively thwart the autophagic pathway.
Highlights
Penetration of brain endothelium by Group B streptococcus (GBS) is the first step in the development of meningitis
Our results demonstrate that GBS infection triggers a robust autophagic response in brain endothelium and that this response contributes to limiting intracellular bacteria
We investigated the turnover of the autophagic adaptor protein p62/sequestome-1 [42], which interacts with light chain 3 (LC3) and is an indicator of active autophagy [43,44,45,46,47]
Summary
Penetration of brain endothelium by Group B streptococcus (GBS) is the first step in the development of meningitis. Structures even though we observed GBS-LC3 co-localization These results suggest that autophagy may act as a BBB cellular defense mechanism in response to invading and toxinproducing bacteria, GBS may actively thwart the autophagic pathway. Antimicrobial autophagy, a selective type of autophagy known as xenophagy, has emerged as a potent host defense mechanism against intracellular bacterial and viral pathogens [17, 20] Several pathogenic bacteria such as Salmonella enterica serovar Typhimurium (Salmonella typhimurium), Listeria monocytogenes, Shigella flexneri, and group A Streptococcus (GAS) have been shown to activate the autophagic pathway [21,22,23]. Our studies demonstrate that the GBSsecreted -h/c toxin is sufficient to activate an acute autophagic response in BBB endothelium but that this response may not be adequate to reduce the majority of intracellular GBS
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