Dataset Information

Group B Streptococcus interactions with human meningeal cells and astrocytes in vitro.

ABSTRACT:

Background

Streptococcus agalactiae (Group B Streptococcus, GBS) is a leading cause of life-threatening neonatal meningitis and survivors often suffer permanent neurological damage. How this organism interacts with the meninges and subsequently with astrocytes that constitute the underlying cortical glia limitans superficialis is not known.

Methodology/principal findings

In this paper, we demonstrate dose-dependent adherence of GBS over time to human meningioma cells and fetal astrocytes in vitro, which was not influenced by expression of either ?-haemolysin/cytolysin (?-h/c) toxin, different capsule serotypes or by absence of capsule (p>0.05). Internalization of GBS by both cell types was, however, a slow and an infrequent event (only 0.02-0.4% of associated bacteria were internalised by 9 h). Expression of ?-h/c toxin did not play a role in invasion (p>0.05), whereas capsule expression lead to a reduction (p<0.05) in the numbers of intracellular bacteria recovered. GBS strains induced cytotoxicity as demonstrated by the measurement of lactate dehydrogenase (LDH) enzyme release by 9 h and by viable staining. Increasing levels of meningioma cell death correlated with bacterial growth and the phenotype of ?-h/c toxin production, i.e. from weakly, to normo- to hyper-haemolytic. However, cytotoxicity was significantly greater (p<0.05) towards astrocytes, and infection with initial MOI?0.003 induced 70-100% LDH release. By comparing wild-type (?-h/c(+)) and mutant (?cylE ?-h/c(-)) strains and ?-h/c toxin extracts and by using the surfactant dipalmitoylphosphatidylcholine in cytotoxicity inhibition experiments, ?-h/c toxin was demonstrated as principally responsible for cell death.

Conclusions/significance

This study has described key events in the interactions of GBS with meningeal cells and astrocytes in vitro and a major virulence role for ?-h/c toxin. Understanding the mechanisms involved will help to identify potential therapies for improving patient survival and for reducing the incidence and severity of neurological sequelae.

SUBMITTER: Alkuwaity K

PROVIDER: S-EPMC3416839 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Publications

Group B Streptococcus interactions with human meningeal cells and astrocytes in vitro.

Alkuwaity Khalil K Taylor Alexander A Heckels John E JE Doran Kelly S KS Christodoulides Myron M

PloS one 20120810 8

<h4>Background</h4>Streptococcus agalactiae (Group B Streptococcus, GBS) is a leading cause of life-threatening neonatal meningitis and survivors often suffer permanent neurological damage. How this organism interacts with the meninges and subsequently with astrocytes that constitute the underlying cortical glia limitans superficialis is not known.<h4>Methodology/principal findings</h4>In this paper, we demonstrate dose-dependent adherence of GBS over time to human meningioma cells and fetal ast ...[more]

PMID: 22900037

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Project description:BackgroundStreptococcus gallolyticus subsp. gallolyticus is an important causative agent of infective endocarditis (IE) but the knowledge on virulence factors is limited and the pathogenesis of the infection is poorly understood. In the present study, we established an experimental in vitro IE cell culture model using EA.hy926 and HUVEC cells to investigate the adhesion and invasion characteristics of 23 Streptococcus gallolyticus subsp. gallolyticus strains from different origins (human IE-derived isolates, other human clinical isolates, animal isolates). Adhesion to eight components of the extracellular matrix (ECM) and the ability to form biofilms in vitro was examined in order to reveal features of S. gallolyticus subsp. gallolyticus endothelial infection. In addition, the strains were analyzed for the presence of the three virulence factors gtf, pilB, and fimB by PCR.ResultsThe adherence to and invasion characteristics of the examined S. gallolyticus subsp. gallolyticus strains to the endothelial cell line EA.hy926 differ significantly among themselves. In contrast, the usage of three different in vitro models (EA.hy926 cells, primary endothelial cells (HUVECs), mechanical stretched cells) revealed no differences regarding the adherence to and invasion characteristics of different strains. Adherence to the ECM proteins collagen I, II and IV revealed the highest values, followed by fibrinogen, tenascin and laminin. Moreover, a strong correlation was observed in binding to these proteins by the analyzed strains. All strains show the capability to adhere to polystyrole surfaces and form biofilms. We further confirmed the presence of the genes of two known virulence factors (fimB: all strains, gtf: 19 of 23 strains) and demonstrated the presence of the gene of one new putative virulence factor (pilB: 9 of 23 strains) by PCR.ConclusionOur study provides the first description of S. gallolyticus subsp. gallolyticus adhesion and invasion of human endothelial cells, revealing important initial information of strain variability, behaviour and characteristics of this as yet barely analyzed pathogen.

Project description:UnlabelledAs key components of innate immune defense, macrophages are essential in controlling bacterial pathogens, including group A Streptococcus(GAS). Despite this, only a limited number of studies have analyzed the recovery of GAS from within human neutrophils and macrophages. Here, we determined the intracellular fate of GAS in human macrophages by using several quantitative approaches. In both U937 and primary human macrophages, the appearance over time of long GAS chains revealed that despite GAS-mediated cytotoxicity, replication occurred in viable, propidium iodide-negative macrophages. Whereas the major virulence factor M1 did not contribute to bacterial growth, a GAS mutant strain deficient in streptolysin O (SLO) was impaired for intracellular replication. SLO promoted bacterial escape from the GAS-containing vacuole (GCV) into the macrophage cytosol. Up to half of the cytosolic GAS colocalized with ubiquitin and p62, suggesting that the bacteria were targeted by the autophagy machinery. Despite this, live imaging of U937 macrophages revealed proficient replication of GAS after GCV rupture, indicating that escape from the GCV is important for growth of GAS in macrophages. Our results reveal that GAS can replicate within viable human macrophages, with SLO promoting GCV escape and cytosolic growth, despite the recruitment of autophagy receptors to bacteria.ImportanceClassically regarded as an extracellular pathogen, GAS can persist within human epithelial cells, as well as neutrophils and macrophages. Some studies suggest that GAS can modulate its intracellular vacuole to promote survival and perhaps replicate in macrophages. However, an in-depth single-cell analysis of the dynamics of survival and replication is lacking. We used macrophage-like cell lines and primary macrophages to measure the intracellular growth of GAS at both the population and single-cell levels. While CFU counts revealed no increase in overall bacterial growth, quantitative fluorescence microscopy, flow cytometry, and time-lapse imaging revealed bacterial replication in a proportion of infected macrophages. This study emphasizes the importance of single-cell analysis especially when studying the intracellular fate of a pathogen that is cytotoxic and displays heterogeneity in terms of intracellular killing and growth. To our knowledge, this study provides the first direct visualization of GAS replication inside human cells.

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Dataset Information

Group B Streptococcus interactions with human meningeal cells and astrocytes in vitro.

Background

Methodology/principal findings

Conclusions/significance

Publications

Group B Streptococcus interactions with human meningeal cells and astrocytes in vitro.

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