The Streptococcus pneumoniae transcriptome in patient cerebrospinal fluid identifies novel virulence factors required for meningitis

Abstract

To better understand Streptococcus pneumoniae pathogenesis we performed RNA sequencing on cerebrospinal fluid (CSF) from meningitis patients to identify bacterial genes expressed during invasion of the central nervous system. Comparison to transcriptome data for serotype 1 S. pneumoniae cultured in ex vivo human CSF defined a subset of 57 genes with high expression during human meningitis. Deletion of two of the most highly expressed genetic loci, bgaA (encodes for a beta-galactosidase) or the SP_1801-5 putative stress response operon, resulted in S. pneumoniae strains still able to transmigrate the blood brain barrier but which were more susceptible to complement opsonisation and unable to maintain brain infection in a murine meningitis model. In 1144 meningitis patients, infection with bgaA containing S. pneumoniae strains was associated with a higher mortality (22% versus 14% p=0.02). These data demonstrate that direct bacterial RNAseq from CSF can identify previously undescribed S. pneumoniae virulence factors required for meningitis pathogenesis.

The CSF transcriptome in pneumococcal meningitis reveals compartmentalised host inflammatory responses associated with mortality

“…Across samples, between 41-91% of cells were determined to be neutrophils (Figure 2). As an additional control for the CIBERSORTx analysis, we included two available transcriptomes from S. pneumoniae cultured in vitro CSF in the presence of freshly purified neutrophils in the analysis 34 , finding that CIBERSORTx correctly identified these transcriptomes as primary neutrophils. Other cell types detected in the PM-CSF samples included an unexpectedly high proportion of activated mast cells (up to 40% of cells / sample).…”

“…RNA was extracted following a method developed by Mann et al, using the MirVana phenol based extraction kit (Thermofisher) as previous reported as part of a protocol to investigate the in vitro pneumococcal transcriptome. 34,95 Briefly, samples were thawed, pelleted and RNA protection media was removed. Cell lysis buffer was applied, samples were placed in a FastPrep MatrixE tube, undergoing mechanical cell wall disruption in a Precellys machine speed of 6200 rpm for 45 seconds.…”

“…Ribosomal RNA was depleted using human rRNA depletion beads (NewEnglandBiolabs). Libraries for all samples were prepared simultaneously to minimise batch effects as previously 34 .…”