In vivo proteomics identifies the competence regulon and AliB oligopeptide transporter as pathogenic factors in pneumococcal meningitis

Abstract

Streptococcus pneumoniae (pneumococci) is a leading cause of severe bacterial meningitis in many countries worldwide. To characterize the repertoire of fitness and virulence factors predominantly expressed during meningitis we performed niche-specific analysis of the in vivo proteome in a mouse meningitis model, in which bacteria are directly inoculated into the cerebrospinal fluid (CSF) cisterna magna. We generated a comprehensive mass spectrometry (MS) spectra library enabling bacterial proteome analysis even in the presence of eukaryotic proteins. We recovered 200,000 pneumococci from CSF obtained from meningitis mice and by MS we identified 685 pneumococci proteins in samples from in vitro filter controls and 249 in CSF isolates. Strikingly, the regulatory two-component system ComDE and substrate-binding protein AliB of the oligopeptide transporter system were exclusively detected in pneumococci recovered from the CSF. In the mouse meningitis model, AliB-, ComDE-, or AliB-ComDE-deficiency resulted in attenuated meningeal inflammation and disease severity when compared to wild-type pneumococci indicating the crucial role of ComDE and AliB in pneumococcal meningitis. In conclusion, we show here mechanisms of pneumococcal adaptation to a defined host compartment by a proteome-based approach. Further, this study provides the basis of a promising strategy for the identification of protein antigens critical for invasive disease caused by pneumococci and other meningeal pathogens.

CSF Levels of Elongation Factor Tu Is Associated With Increased Mortality in Malawian Adults With Streptococcus pneumoniae Meningitis

“…The number and range of S. pneumoniae proteins identified in the CSF were similar to our earlier study, including PspA, abundant ribosomal proteins and EF-Tu (Gomez-Baena et al, 2017). These proteins differ from those identified in a CSF proteome study in a murine model of PM (Schmidt et al, 2019), in which high CSF levels of the competence regulator ComDE and the AliB oligopeptide transporter in murine meningitis were associated with leukocyte recruitment to the CSF compartment and disease severity (Schmidt et al, 2019). However, we did not detect either expression of ComDE or AliB in patient PM CSF, and we also did not detect other, previously reported virulence proteins in PM including pneumolysin (Wall et al, 2012;Wippel et al, 2013).…”

Pneumolysin induces platelet destruction, not platelet activation, which can be prevented by immunoglobulin preparations in vitro

“…13 Briefly, we incubated washed human platelets in Tyrode buffer containing Ca 21 and Mg 21 with phosphate-buffered saline (PBS), 20 mM thrombin receptor activator peptide 6 (TRAP-6), pneumolysin, or pneumococci (D39, TIGR4, or their pneumolysin-free mutants D39Dply and TIGR4Dply). 14,15 We did grow bacteria to the mid-log exponential phase before incubating 1.8 3 10 6 bacteria with 9 3 10 6 platelets (ratio bacteria platelets 1:5) for 2 or 3 hours. We incubated platelets with pneumolysin at different concentrations or the pneumolysin mutants for 10 minutes.…”

“…We determined cytolytic activity of purified pneumolysin proteins by the hemolysis activity test as described. 14 In brief, we incubated ACD-A blood from healthy human volunteers with pneumolysin WT , pneumolysin C428G , and pneumolysin W433F for 10 minutes at 37°C in a 96-well plate (U-bottom). After incubation, we centrifuged the plate and monitored formation of the erythrocyte sediment.…”

“…Experiments were performed according to International Society on Thrombosis and Haemostasis Scientific and Standardization Committee (ISTH SSC) subcommittee on Biorheology recommendations. 14…”

Virulence Factors of Meningitis-Causing Bacteria: Enabling Brain Entry across the Blood–Brain Barrier

“…Furthermore, choline binding protein PspC was shown to bind only to pIgR [97], and interaction with the laminin receptor by S. pneumoniae is initiated by choline-binding protein A (CbpA) [99]. A proteome-based approach in a mouse meningitis model, addressing adaptive capabilities of the pathogens to a defined host compartment, has highlighted a crucial role for two highly expressed pneumococcal proteins; ComDE, a regulatory two-component system, and AliB, a substrate-binding protein of an oligopeptide transporter, in pneumococcal meningitis [100].…”