Strategies to Isolate Extracellular Vesicles from Gram-Negative and Gram-Positive Bacteria.

Membrane vesicles are produced by all Gram-negative and Gram-positive bacteria investigated so far. Membrane vesicles are spherical bilayers of phospholipids released by the bacteria to their surrounding environment and whose average size is comprised between 20 and 300 nm. The purification of these vesicles is often a challenge, as the yield and purity are often crucial for further analyses or use. In this chapter, we describe the most used method to isolate membrane vesicles from culture supernatant of Streptococcus pneumoniae and Klebsiella pneumoniae using ultracentrifugation followed by a density gradient method.

Membrane particles evoke a serotype-independent cross-protection against pneumococcal infection that is dependent on the conserved lipoproteins MalX and PrsA.

Pneumococcal conjugate vaccines (PCVs) used in childhood vaccination programs have resulted in replacement of vaccine-type with nonvaccine-type pneumococci in carriage and invasive pneumococcal disease (IPD). A vaccine based on highly conserved and protective pneumococcal antigens is urgently needed. Here, we performed intranasal immunization of mice with pneumococcal membrane particles (MPs) to mimic natural nasopharyngeal immunization. MP immunization gave excellent serotype-independent protection against IPD that was antibody dependent but independent of the cytotoxin pneumolysin. Using Western blotting, immunoprecipitation, mass spectrometry, and different bacterial mutants, we identified the conserved lipoproteins MalX and PrsA as the main antigens responsible for cross-protection. Additionally, we found that omitting the variable surface protein and vaccine candidate PspA from MPs enhanced protective immune responses to the conserved proteins. Our findings suggest that MPs containing MalX and PrsA could serve as a platform for pneumococcal vaccine development targeting the elderly and immunocompromised.

Encapsulation of the septal cell wall protects Streptococcus pneumoniae from its major peptidoglycan hydrolase and host defenses.

Synthesis of the capsular polysaccharide, a major virulence factor for many pathogenic bacteria, is required for bacterial survival within the infected host. In Streptococcus pneumoniae, Wze, an autophosphorylating tyrosine kinase, and Wzd, a membrane protein required for Wze autophosphorylation, co-localize at the division septum and guarantee the presence of capsule at this subcellular location. To determine how bacteria regulate capsule synthesis, we studied pneumococcal proteins that interact with Wzd and Wze using bacterial two hybrid assays and fluorescence microscopy. We found that Wzd interacts with Wzg, the putative ligase that attaches capsule to the bacterial cell wall, and recruits it to the septal area. This interaction required residue V56 of Wzd and both the transmembrane regions and DNA-PPF domain of Wzg. When compared to the wild type, Wzd null pneumococci lack capsule at midcell, bind the peptidoglycan hydrolase LytA better and are more susceptible to LytA-induced lysis, and are less virulent in a zebrafish embryo infection model. In this manuscript, we propose that the Wzd/Wze pair guarantees full encapsulation of pneumococcal bacteria by recruiting Wzg to the division septum, ensuring that capsule attachment is coordinated with peptidoglycan synthesis. Impairing the encapsulation process, at localized subcellular sites, may facilitate elimination of bacteria by strategies that target the pneumococcal peptidoglycan.

A molecular link between cell wall biosynthesis, translation fidelity, and stringent response in Streptococcus pneumoniae.

Survival in the human host requires bacteria to respond to unfavorable conditions. In the important Gram-positive pathogen Streptococcus pneumoniae, cell wall biosynthesis proteins MurM and MurN are tRNA-dependent amino acyl transferases which lead to the production of branched muropeptides. We demonstrate that wild-type cells experience optimal growth under mildly acidic stressed conditions, but ΔmurMN strain displays growth arrest and extensive lysis. Furthermore, these stress conditions compromise the efficiency with which alanyl-tRNAAla synthetase can avoid noncognate mischarging of tRNAAla with serine, which is toxic to cells. The observed growth defects are rescued by inhibition of the stringent response pathway or by overexpression of the editing domain of alanyl-tRNAAla synthetase that enables detoxification of tRNA misacylation. Furthermore, MurM can incorporate seryl groups from mischarged Seryl-tRNAAlaUGC into cell wall precursors with exquisite specificity. We conclude that MurM contributes to the fidelity of translation control and modulates the stress response by decreasing the pool of mischarged tRNAs. Finally, we show that enhanced lysis of ΔmurMN pneumococci is caused by LytA, and the murMN operon influences macrophage phagocytosis in a LytA-dependent manner. Thus, MurMN attenuates stress responses with consequences for host-pathogen interactions. Our data suggest a causal link between misaminoacylated tRNA accumulation and activation of the stringent response. In order to prevent potential corruption of translation, consumption of seryl-tRNAAla by MurM may represent a first line of defense. When this mechanism is overwhelmed or absent (ΔmurMN), the stringent response shuts down translation to avoid toxic generation of mistranslated/misfolded proteins.

Neuronal death in pneumococcal meningitis is triggered by pneumolysin and RrgA interactions with ß-actin.

Neuronal damage is a major consequence of bacterial meningitis, but little is known about mechanisms of bacterial interaction with neurons leading to neuronal cell death. Streptococcus pneumoniae (pneumococcus) is a leading cause of bacterial meningitis and many survivors develop neurological sequelae after the acute infection has resolved, possibly due to neuronal damage. Here, we studied mechanisms for pneumococcal interactions with neurons. Using human primary neurons, pull-down experiments and mass spectrometry, we show that pneumococci interact with the cytoskeleton protein ß-actin through the pilus-1 adhesin RrgA and the cytotoxin pneumolysin (Ply), thereby promoting adhesion and invasion of neurons, and neuronal death. Using our bacteremia-derived meningitis mouse model, we observe that RrgA- and Ply-expressing pneumococci co-localize with neuronal ß-actin. Using purified proteins, we show that Ply, through its cholesterol-binding domain 4, interacts with the neuronal plasma membrane, thereby increasing the exposure on the outer surface of ß-actin filaments, leading to more ß-actin binding sites available for RrgA binding, and thus enhanced pneumococcal interactions with neurons. Pneumococcal infection promotes neuronal death possibly due to increased intracellular Ca2+ levels depending on presence of Ply, as well as on actin cytoskeleton disassembly. STED super-resolution microscopy showed disruption of ß-actin filaments in neurons infected with pneumococci expressing RrgA and Ply. Finally, neuronal death caused by pneumococcal infection could be inhibited using antibodies against ß-actin. The generated data potentially helps explaining mechanisms for why pneumococci frequently cause neurological sequelae.