Chiara Tani

Expression of factor H binding protein in meningococcal strains can vary at least 15-fold and is genetically determined

Significance Complement is the main line of defense against bacterial pathogens; however, the molecular mechanisms triggering killing are largely unknown. Factor H binding protein (fHbp) is a component of two licensed vaccines against serogroup B meningococcus and a key target of complement-mediated bacterial killing. Selected reaction monitoring was used for the absolute quantification of fHbp on invasive meningococcal strains, showing that expression among strains can vary at least 15-fold and a minimum of 757 molecules separated by not more than 130 nm are required to engage C1q and kill the bacteria. Furthermore, the amount of fHbp is genetically determined by the sequence of the promoter region and correlated with the bactericidal activity. These findings increase the understanding of complement-mediated killing and vaccine protection. 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.

Quantification by LC–MSE of outer membrane vesicle proteins of the Bexsero® vaccine

Meningococcal disease is a major cause of morbidity and mortality worldwide. Its epidemiology is currently dominated by five capsular serogroups (A, B, C, W, and Y). While effective vaccines already exist for serogroups A, C, W and Y, except for under clonal outbreaks, no vaccine was available against serogroup B. Recently, a four component vaccine, Bexsero(®), designed to prevent infection caused by this serogroup, has been approved in Europe and other Countries for use in individuals from two months of age and older. The active components of this vaccine are three recombinant proteins identified by reverse vaccinology combined with detergent extracted outer membrane vesicles (DOMV) prepared from a New Zealand epidemic strain. Considering their intrinsic complexity, we performed additional characterization of DOMVs on top of the standard quality control testing carried out for batch release. We applied the Hi3 label-free LC-MS(E) methodology to qualitatively and quantitatively characterize the DOMV protein content. We first, successfully investigated the robustness and the accuracy of the methodology for the DOMV characterization and we then applied it to compare six DOMV production lots. Around 100 proteins were quantified from each preparation. When classified according to their predicted cellular localization, about 90% of the total protein amount belongs consistently to the outer membrane compartment. Using nonparametric hypothesis testing and complementary log-log linear regression, the quantifications of a subset of 21 proteins common to all lots and including approximately 90% (85-92%) of the total protein amount quantified in any DOMV lot were found consistent across lots. The relevance of these results is two-fold, showing that the Hi3 quantification methodology is robust for a broad range of proteins and indicating that the manufacturing process currently used for the production of the Bexsero(®) DOMV components is highly reproducible and consistent.

Multidisciplinary Analysis of a Nontoxigenic Clostridium difficile Strain with Stable Resistance to Metronidazole

ABSTRACT Stable resistance to metronidazole in a nontoxigenic Clostridium difficile strain was investigated at both the genomic and proteomic levels. Alterations in the metabolic pathway involving the pyruvate-ferredoxin oxidoreductase were found, suggesting that reduction of metronidazole, required for its activity, may be less efficient in this strain. Proteomic studies also showed a cellular response to oxidative stress.

LytM Proteins Play a Crucial Role in Cell Separation, Outer Membrane Composition, and Pathogenesis in Nontypeable Haemophilus influenzae

ABSTRACT LytM proteins belong to a family of bacterial metalloproteases. In Gram-negative bacteria, LytM factors are mainly reported to have a direct effect on cell division by influencing cleavage and remodeling of peptidoglycan. In this study, mining nontypeable Haemophilus influenzae (NTHI) genomes, three highly conserved open reading frames (ORFs) containing a LytM domain were identified, and the proteins encoded by the ORFs were named YebA, EnvC, and NlpD on the basis of their homology with the Escherichia coli proteins. Immunoblotting and confocal analysis showed that while NTHI NlpD is exposed on the bacterial surface, YebA and EnvC reside in the periplasm. NTHI ΔyebA and ΔnlpD deletion mutants revealed an aberrant division phenotype characterized by an altered cell architecture and extensive membrane blebbing. The morphology of the ΔenvC deletion mutant was identical to that of the wild-type strain, but it showed a drastic reduction of periplasmic proteins, including the chaperones HtrA, SurA, and Skp, and an accumulation of β-barrel-containing outer membrane proteins comprising the autotransporters Hap, IgA serine protease, and HMW2A, as observed by proteomic analysis. These data suggest that EnvC may influence the bacterial surface protein repertoire by facilitating the passage of the periplasmic chaperones through the peptidoglycan layer to the close vicinity of the inner face of the outer membrane. This hypothesis was further corroborated by the fact that an NTHI envC defective strain had an impaired capacity to adhere to epithelial cells and to form biofilm. Notably, this strain also showed a reduced serum resistance. These results suggest that LytM factors are not only important components of cell division but they may also influence NTHI physiology and pathogenesis by affecting membrane composition. IMPORTANCE Nontypeable Haemophilus influenzae (NTHI) is an opportunistic pathogen that colonizes the human nasopharynx and can cause serious infections in children (acute otitis media) and adults (chronic obstructive pulmonary disease). Several virulence factors are well studied, but the complete scenario of NTHI pathogenesis is still unclear. We identified and characterized three NTHI LytM factors homologous to the Escherichia coli LytM proteins. Although LytM factors are reported to play a crucial role in the cell division process, in NTHI they are also involved in other bacterial functions. In particular, YebA and NlpD are fundamental for membrane stability: indeed, their absence causes an increased release of outer membrane vesicles (OMVs). On the other hand, our data suggest that EnvC could directly or indirectly affect peptidoglycan permeability and consequently, bacterial periplasmic and outer membrane protein distribution. Interestingly, by modulating the surface composition of virulence determinants, EnvC also has an impact on NTHI pathogenesis. Nontypeable Haemophilus influenzae (NTHI) is an opportunistic pathogen that colonizes the human nasopharynx and can cause serious infections in children (acute otitis media) and adults (chronic obstructive pulmonary disease). Several virulence factors are well studied, but the complete scenario of NTHI pathogenesis is still unclear. We identified and characterized three NTHI LytM factors homologous to the Escherichia coli LytM proteins. Although LytM factors are reported to play a crucial role in the cell division process, in NTHI they are also involved in other bacterial functions. In particular, YebA and NlpD are fundamental for membrane stability: indeed, their absence causes an increased release of outer membrane vesicles (OMVs). On the other hand, our data suggest that EnvC could directly or indirectly affect peptidoglycan permeability and consequently, bacterial periplasmic and outer membrane protein distribution. Interestingly, by modulating the surface composition of virulence determinants, EnvC also has an impact on NTHI pathogenesis.

Differential response to intracellular stress in the skin from osteogenesis imperfecta Brtl mice with lethal and non lethal phenotype: A proteomic approach☆

Phenotypic variability in the presence of an identical molecular defect is a recurrent feature in heritable disorders and it was also reported in osteogenesis imperfecta (OI). OI is a prototype for skeletal dysplasias mainly caused by mutations in the two genes coding for type I collagen. No definitive cure is available for this disorder, but the understanding of molecular basis in OI phenotypic modulation will have a pivotal role in identifying possible targets to develop novel drug therapy. We used a functional proteomic approach to address the study of phenotypic variability using the skin of the OI murine model Brtl. Brtl mice reproduce the molecular defect, dominant transmission and phenotypic variability of human OI patients. In the presence of a Gly349Cys substitution in α1(I)-collagen Brtl mice can have a lethal or a moderately severe outcome. Differential expression of chaperones, proteasomal subunits, metabolic enzymes, and proteins related to cellular fate demonstrated that a different ability to adapt to cellular stress distinguished mutant from wild-type mice and mutant lethal from surviving mutant animals. Interestingly, class discovery analysis identified clusters of differentially expressed proteins associated with a specific outcome, and functional analysis contributed to a deeper investigation into biochemical and cellular pathways affected by the disease. This article is part of a Special Issue entitled: Translational Proteomics.

The human pathogen Streptococcus pyogenes releases lipoproteins as Lipoprotein-rich Membrane Vesicles

Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed 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.

Generation and characterization of a bivalent protein boost for future clinical trials: HIV-1 subtypes CR01_AE and B gp120 antigens with a potent adjuvant

The RV144 Phase III clinical trial with ALVAC-HIV prime and AIDSVAX B/E subtypes CRF01_AE (A244) and B (MN) gp120 boost vaccine regime in Thailand provided a foundation for the future development of improved vaccine strategies that may afford protection against the human immunodeficiency virus type 1 (HIV-1). Results from this trial showed that immune responses directed against specific regions V1V2 of the viral envelope (Env) glycoprotein gp120 of HIV-1, were inversely correlated to the risk of HIV-1 infection. Due to the low production of gp120 proteins in CHO cells (2–20 mg/L), cleavage sites in V1V2 loops (A244) and V3 loop (MN) causing heterogeneous antigen products, it was an urgent need to generate CHO cells harboring A244 gp120 with high production yields and an additional, homogenous and uncleaved subtype B gp120 protein to replace MN used in RV144 for the future clinical trials. Here we describe the generation of Chinese Hamster Ovary (CHO) cell lines stably expressing vaccine HIV-1 Env antigens for these purposes: one expressing an HIV-1 subtype CRF01_AE A244 Env gp120 protein (A244.AE) and one expressing an HIV-1 subtype B 6240 Env gp120 protein (6240.B) suitable for possible future manufacturing of Phase I clinical trial materials with cell culture expression levels of over 100 mg/L. The antigenic profiles of the molecules were elucidated by comprehensive approaches including analysis with a panel of well-characterized monoclonal antibodies recognizing critical epitopes using Biacore and ELISA, and glycosylation analysis by mass spectrometry, which confirmed previously identified glycosylation sites and revealed unknown sites of O-linked and N-linked glycosylations at non-consensus motifs. Overall, the vaccines given with MF59 adjuvant induced higher and more rapid antibody (Ab) responses as well as higher Ab avidity than groups given with aluminum hydroxide. Also, bivalent proteins (A244.AE and 6240.B) formulated with MF59 elicited distinct V2-specific Abs to the epitope previously shown to correlate with decreased risk of HIV-1 infection in the RV144 trial. All together, these results provide critical information allowing the consideration of these candidate gp120 proteins for future clinical evaluations in combination with a potent adjuvant.