Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.MenB vaccine | selected reaction monitoring mass spectrometry | SRM-MS | fHbp antigen | antigen density

SpyCEP is a 170-kDa multidomain serine protease expressed on the surface of the human pathogen Streptococcus pyogenes, which plays an important role in infection by catalyzing cleavage and inactivation of the neutrophil chemoattractant interleukin-8. In this study, we investigated the biochemical features and maturation process of SpyCEP, starting from a recombinant form of the protease expressed and purified from Escherichia coli. We show that active recombinant SpyCEP differs from other bacterial proteases in that it is constituted by 2 noncovalently linked fragments derived from autocatalytic processing, an N-terminal fragment of 210 aa bearing one of the 3 catalytic triad residues, and a 1369-residue C-terminal polypeptide containing the remaining 2 catalytic amino acids. The same type of organization is present in the enzyme obtained from S. pyogenes. Furthermore, N-terminal SpyCEP is not involved in the folding of the mature enzyme. The 2 protease fragments were separately expressed in E. coli as soluble polypeptides that, when combined, reconstituted a fully active enzyme complex. Therefore, SpyCEP appears to possess a completely new structural architecture that has not been described so far for other microbial proteases.

Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surfaceexposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles. Molecular & Cellular

This study used proteomic and transcriptomic techniques to understand the molecular basis of the phenotypic variability in the bone disorder osteogenesis imperfecta (OI). Calvarial bone mRNA expression was evaluated by microarray, real-time, and comparative RT-PCR and the bone proteome profile was analyzed by 2-DE, MS, and immunoblotting in the OI murine model BrtlIV, which has either a moderate or a lethal OI outcome. Differential expression analysis showed significant changes for eight proteins. The expression of the ER stress-related protein Gadd153 was increased in lethal mice, whereas expression of the chaperone alphaB crystallin was increased in nonlethal mice, suggesting that the intracellular machinery is involved in the modulation of the OI phenotype. Furthermore, in lethal BrtlIV, the increased expression of the cartilaginous proteins Prelp, Bmp6, and Bmp7 and the lower expression of the bone matrix proteins matrilin 4, microfibril-associated glycoprotein 2, and thrombospondin 3 revealed that both a delay in skeletal development and an alteration in extracellular matrix composition influence OI outcomes. Differentially expressed proteins identified in this model offer a starting point for elucidating the molecular basis of phenotypic variability, a characteristic common to many genetic disorders. The first reference 2-DE map for murine calvarial tissue is also reported.