Cysteine Depletion Causes Oxidative Stress and Triggers Outer Membrane Vesicle Release by Neisseria meningitidis; Implications for Vaccine Development

Abstract

Outer membrane vesicles (OMV) contain immunogenic proteins and contribute to in vivo survival and virulence of bacterial pathogens. The first OMV vaccines successfully stopped Neisseria meningitidis serogroup B outbreaks but required detergent-extraction for endotoxin removal. Current vaccines use attenuated endotoxin, to preserve immunological properties and allow a detergent-free process. The preferred process is based on spontaneously released OMV (sOMV), which are most similar to in vivo vesicles and easier to purify. The release mechanism however is poorly understood resulting in low yield. This study with N. meningitidis demonstrates that an external stimulus, cysteine depletion, can trigger growth arrest and sOMV release in sufficient quantities for vaccine production (±1500 human doses per liter cultivation). Transcriptome analysis suggests that cysteine depletion impairs iron-sulfur protein assembly and causes oxidative stress. Involvement of oxidative stress is confirmed by showing that addition of reactive oxygen species during cysteine-rich growth also triggers vesiculation. The sOMV in this study are similar to vesicles from natural infection, therefore cysteine-dependent vesiculation is likely to be relevant for the in vivo pathogenesis of N. meningitidis.

Interspecies Communication between Pathogens and Immune Cells via Bacterial Membrane Vesicles

“…Interestingly, mutants of oxidative stress response pathways correlated with a hypovesiculation phenotype. This finding is consistent with the observation that MV production is upregulated in response to oxidative stress (Sabra et al, 2003; MacDonald and Kuehn, 2013; van de Waterbeemd et al, 2013). MVs are secreted constitutively by bacteria, but this process is also induced by iron-deprivation, antimicrobial peptides, and envelope stress (Manning and Kuehn, 2011; MacDonald and Kuehn, 2013; Prados-Rosales et al, 2014b).…”

Efficient quantification and characterization of bacterial outer membrane derived nano-particles with flow cytometric analysis

“…For the first time this would also allow for experiments based on multiplicity of infection (MOI) regarding OMVs and eukaryotic or bacterial cells. OMV production rates of bacteria are also unclear so far and might vary considerably, the same is true for vaccination doses based on purified OMVs (Kulp and Kuehn, 2010;Van de Waterbeemd et al, 2013). Thus, accurate and efficient enumeration of OMVs is an extremely desirable goal (Berleman and Auer, 2013;Camacho et al, 2013;Ellis and Kuehn, 2010;Schaar et al, 2013;Shen et al, 2012;Wai et al, 2003;Zhao et al, 2013).…”

Comparative Proteome Analysis of Spontaneous Outer Membrane Vesicles and Purified Outer Membranes of Neisseria meningitidis

“…Oxidative stress triggers vesiculation (7), which might be a mechanism to shed OM contaminated with antimicrobials or misfolded proteins (6). So far, no conserved mechanisms of bacterial vesiculation have been defined.…”