The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence

Abstract

GNA2091 is one of the components of the 4‐component meningococcal serogroup B vaccine (4CMenB) vaccine and is highly conserved in all meningococcal strains. However, its functional role has not been fully characterized. Here we show that nmb2091 is part of an operon and is cotranscribed with the nmb2089, nmb2090, and nmb2092 adjacent genes, and a similar but reduced operon arrangement is conserved in many other gram‐negative bacteria. Deletion of the nmb2091 gene causes an aggregative phenotype with a mild defect in cell separation; differences in the outer membrane composition and phospholipid profile, in particular in the phosphoethanolamine levels; an increased level of outer membrane vesicles; and deregulation of the zinc‐responsive genes such as znuD. Finally, the Δ2091 strain is attenuated with respect to the wild‐type strain in competitive index experiments in the infant rat model of meningococcal infection. Altogether these data suggest that GNA2091 plays important roles in outer membrane architecture, biogenesis, homeostasis, and in meningococcal survival in vivo, and amodel for its role is discussed. These findings highlight the importance of GNA2091 as a vaccine component.—Seib, K. L., Haag, A. F., Oriente, F., Fantappiè, L., Borghi, S., Semchenko, E. A., Schulz, B. L., Ferlicca, F., Taddei, A. R., Giuliani, M. M., Pizza, M., Delany, I. The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence. FASEB J. 33, 12324‐12335 (2019). http://www.fasebj.org

Outer membrane lipoprotein DolP interacts with the BAM complex and promotes fitness during envelope stress response

“…The MG1655 derivative strain LC-E75, harbouring a dCas9-encoding construct under the control of the P tet promoter, has been described 38 . Cells were cultured in home-made lysogeny broth (LB) medium (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 5 mg/ml (NaCl), commercially available Miller LB Broth (Sigma) or M9 minimal medium containing M9 salts (33.7 mM Na 2 HPO 4 , 22 mM KH 2 PO 4 , 8.55 mM NaCl, 9.35 mM NH 4 Cl) and supplemented with 0.2% w/v glycerol and all the amino acids, or all amino acids except methionine and cysteine in the case of 35 S pulse-chase labeling. Antibiotics were used at the following concentrations: ampicillin 100 μg/ml, kanamycin 50 μg/ml, vancomycin 60 μg/ml.…”

“…Pulse-chase labeling of cellular proteins with radioactive 35 S methionine and cysteine was conducted as previously described 79 . Briefly, overnight cultures were washed and freshly diluted into M9 medium to OD 600 = 0.03 and incubated at 37 °C.…”

“…When the cultures reached OD 600 = 0.3, ectopic expression of EspP was induced by adding 200 μM IPTG for 30 min. 35 S labelling was performed by supplementing cultures with a mixture of [ 35 S]-methionine and [ 35 S]-cysteine followed after 30 seconds by the addition of an excess of cold methionine and cysteine. Samples were withdrawn at different chase times, immediately mixed with an equal volume of ice and subjected to TCA precipitation, prior to immunoprecipitation.…”

“…Thus, the proteolytic reaction can be exploited to infer about completion of β-barrel folding and assembly 82 . To monitor the kinetics of EspP autoproteolysis, cells carrying pRI22 were cultured in M9 minimal medium until early exponential phase, supplemented with 200 μM IPTG and subjected to 35 S pulse-chase labelling. The total protein contents of samples collected after the indicated chase times were subjected to immunoprecipitation using an antiserum specific for an EspP C-terminal peptide.…”

Lipoprotein DolP supports proper folding of BamA in the bacterial outer membrane promoting fitness upon envelope stress

“…Remarkably, the functions of a number of genes upregulated by s E remain unknown. Among those, dolP/yraP (recently renamed division and OM stress-associated lipid-binding protein) encodes a ~20 kDa OM-anchored lipoprotein largely conserved in g and b proteobacteria that is crucial for OM integrity and pathogenicity (Bos et al, 2014, Bryant et al, 2020, Morris et al, 2018, Onufryk et al, 2005, Seib et al, 2019. DolP consists of two consecutive BON (bacterial OsmY and nodulation) domains, a family of conserved folding motifs named after the osmotic stress-induced periplasmic protein OsmY (Yeats & Bateman, 2003).…”