Abstract
Streptococcus pneumoniae is known as an opportunistic pathogen which belongs to the most common etiological agents of local and systemic diseases in humans. In immune compromised patients, pneumococci infiltrate the meninges and cause life threatening inflammation. Recently, we have established a transwell-based Blood Brain Barrier (BBB) model with human endothelial-like cells and astrocytes providing a reliable tool to study pathogenesis mechanisms of bacterial and parasitical transmigration of the cellular barrier between vascular system and cerebral tissue. We applied this model with the aim to study the role of the polysaccharide capsule of a clinical isolate from a meningitis patient identified as a highly virulent serotype 7F pneumococcus. After optimization of the transformation procedure for this clinical isolate, we successfully deleted the capsule gene locus and confirmed loss of capsule expression by southern blot hybridization and electron microscopic visualization. In consistence with the already described inhibiting effect of capsular polysaccharides on phagocytosis by macrophages, the serotype 7F strain was more efficiently endocytosed by human monocytes (U937) as the corresponding capsule deficient mutant. Infection analyses using the BBB model with the serotype 7F wild type isolate and the mutant strain demonstrated a significantly reduced transmigration activity of the capsule-deficient serotype 7F. These results were further confirmed by infection of a second blood brain barrier model using Human Brain Microvascular Endothelial Cells (HBMEC). Interestingly, a collection of serotype 7F strains isolated from cerebral liquor or blood of children suffering from meningitis revealed a higher transmigration capacity as monitored in comparable infection studies with serotype 7F isolate derived from a non-invasive colonization. These data shed new light on the role of pneumococcal capsule in invasive infection process of the cerebral tissues and underline the requirement of adequate infection models to elucidate the diversity of highly virulent clinical isolates.
Highlights
The facultative pathogen Streptococcus pneumoniae is commensally colonizing the upper respiratory epithelium of humans and is described as one of the most important pathogens worldwide [1]
After successful transformation of the clinical serotype 7F-isolate with a DNA sequence comprising the kanamycin cassette flanked with residual sequence stretches from the capsule genes used for homologous recombination, capsule deficient clones were selected
The successful deletion of the capsule gene locus of a serotype 7F meningitis isolate resulted in a significant change of colony morphology and was visualized by transmission electron microscopy after labeling with cationic nanogold particles
Summary
The facultative pathogen Streptococcus pneumoniae is commensally colonizing the upper respiratory epithelium of humans and is described as one of the most important pathogens worldwide [1]. According to the European Center for Disease Prevention and Control (ECDC), up to 14750 cases of invasive pneumococcal diseases in 24 EU and EEA/ EFTA member states were confirmed in 2008. Community-acquired bacterial meningitis remains an ever-present threat, even in developed countries, despite the implementation of childhood vaccination programs and effective antimicrobial agents [4,5]. The most common causes for bacterial meningitis are Streptococcus pneumoniae and Neisseria meningitidis, with the first being responsible for two-thirds of cases in Europe and the United States [4,5]. Despite advances in medical care, mortality from pneumococcal meningitis ranges from 16% to 37% and neurological sequelae including hearing loss, focal neurological deficits, and cognitive impairment, are estimated to occur in up to 52% of surviving patients [6]. During the last decades of years, intensive scientific investigations are focused on the elucidation of pathogenesis mechanisms and virulence factors involved in this invasive infection disease
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