Common-type acylphosphatase is a small cytosolic enzyme whose catalytic properties and three-dime... more Common-type acylphosphatase is a small cytosolic enzyme whose catalytic properties and three-dimensional structure are known in detail. All the acidic residues of the enzyme have been replaced by noncharged residues in order to assess their contributions to the conformational stability of acylphosphatase. The enzymatic activity parameters and the conformational free energy of each mutant were determined by enzymatic activity assays and chemically induced unfolding, respectively. Some mutants exhibit very similar conformational stability, ⌬G(H 2 O), and specific activity values as compared to the wild-type enzyme. By contrast, six mutants show a significant reduction of conformational stability and two mutants are more stable than the wild-type protein. Although none of the mutated acidic residues is directly involved in the catalytic mechanism of the enzyme, our results indicate that mutations of residues located on the surface of the protein are responsible for a structural distortion which propagate up to the active site. We found a good correlation between the free energy of unfolding and the enzymatic activity of acylphosphatase. This suggests that enzymatic activity measurements can provide valuable indications on the conformational stability of acylphosphatase mutants, provided the mutated residue lies far apart from the active site. Moreover, our results indicate that the distortion of hydrogen bonds rather than the loss of electrostatic interactions, contributes to the decrease of the conformational stability of the protein.
Previous experiments suggested that the upstream AUG triplet present in the 5P P untranslated reg... more Previous experiments suggested that the upstream AUG triplet present in the 5P P untranslated region (UTR) of muscle acylphosphatase mRNA is involved in the regulation of protein expression. In this paper, we study the involvement of the 5P PUTR secondary structure and upstream peptide on mRNA stability and protein translation. Our data, obtained using deletion and frame-shift mutants, demonstrate that the 5P PUTR controls protein expression regulating translation together with mRNA stability. Furthermore, we demonstrate that the inhibitory effect of the 5P PUTR of muscle acylphosphatase is relieved during the differentiation process in agreement with previous data reporting an increase of acylphosphatase content during cell differentiation. Finally, UV cross-linking experiments show that specific mRNA-binding proteins are associated with the 5P PUTR of the muscle acylphosphatase mRNA.
The factor H binding protein (fHbp) is a 27-kDa membrane-anchored lipoprotein of Neisseria mening... more The factor H binding protein (fHbp) is a 27-kDa membrane-anchored lipoprotein of Neisseria meningitidis that allows the survival of the bacterium in human plasma; it is also a major component of a universal vaccine against meningococcus B.
International Journal of Medical Microbiology, 2004
With the advent of the genomic era, identification of bacterial factors involved in virulence is ... more With the advent of the genomic era, identification of bacterial factors involved in virulence is a different challenge. Given the vast amount of information available on toxins, in terms of sequence and 3D structure, and thanks to the growing number of sequenced bacterial genomes, it is possible to proceed by homology criteria to predict novel toxins in different microorganisms. ADP-ribosyltransferases constitute a class of functionally conserved enzymes, which display toxic activity in a variety of bacterial pathogens. Since these proteins play a key role in pathogenesis, they have been extensively characterized and successfully used as vaccine components and mucosal adjuvants. Therefore, the application of in silico analyses to identify novel members of this class of enzymes represents an important challenge in the genomic era. To address this subject, different groups have recently pursued homology-based procedures to screen bacterial genomes for novel, yet undiscovered ADP-ribosyltransferases (ADPRTs) and have identified more than twenty novel ADPRTs in Gram-positive and Gram-negative bacteria. We have developed a novel pattern-based computational approach, which, flanked by secondary structure prediction tools, has allowed the identification of previously unrecognised putative ADPRTs. One of them, identified in Neisseria meningitidis has been extensively characterized and shown to possess the predicted enzymatic activity, suggesting a possible role of this protein in the virulence of Meningococcus.
The knowledge of sequences and structures of proteins produced by microbial pathogens is continuo... more The knowledge of sequences and structures of proteins produced by microbial pathogens is continuously increasing. Besides offering the possibility to unravel the mechanisms of pathogenesis at molecular level, structural information provides new tools for vaccine development, such as the opportunity to improve viral and bacterial vaccine candidates by rational design. Structure-based rational design of antigens can optimize the epitope repertoire in terms of accessibility, stability and variability. In the present study, we used epitope mapping information on the well characterized antigen of Neisseria meningitidis factor H binding protein (fHbp) to engineer its gonococcal homologue Ghfp. Meningococcal fHbp is typically classified in three distinct antigenic variants. We introduced epitopes of fHbp variant 1 onto the surface of the Ghfp, which is naturally able to protect against meningococcal strains expressing fHbp of variants 2 and 3. Heterologous epitopes were successfully transplanted, as engineered Ghfp induced functional antibodies against all three fHbp variants. These results confirm that structural vaccinology represents a successful strategy to modulate immune responses as well as a powerful tool to investigate on extension and localization of immuno-dominant epitopes.
Mono ADP-ribosyltransferases (ADPRTs) are a class of functionally conserved enzymes present in pr... more Mono ADP-ribosyltransferases (ADPRTs) are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In bacteria, these enzymes often act as potent toxins and play an important role in pathogenesis. Here we report a profile-based computational approach that, assisted by secondary structure predictions, has allowed the identification of a previously undiscovered ADP-ribosyltransferase in Neisseria meningitidis (NarE). NarE shows structural homologies with E. coli heat-labile enterotoxin (LT) and cholera toxin (CT) and possesses ADP-ribosylating and NAD-glycohydrolase activities. As in the case of LT and CT, NarE catalyses the transfer of the ADP-ribose moiety to arginine residues. Despite the absence of a signal peptide, the protein is efficiently exported into the periplasm of Neisseria. The narE gene is present in 25 out of 43 strains analysed, is always present in ET-5 and Lineage 3 but absent in ET-37 and Cluster A4 hypervirulent lineages. When pre...
Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment... more Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution struc...
Common-type acylphosphatase is a small cytosolic enzyme whose catalytic properties and three-dime... more Common-type acylphosphatase is a small cytosolic enzyme whose catalytic properties and three-dimensional structure are known in detail. All the acidic residues of the enzyme have been replaced by noncharged residues in order to assess their contributions to the conformational stability of acylphosphatase. The enzymatic activity parameters and the conformational free energy of each mutant were determined by enzymatic activity assays
NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens r... more NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens responsible for meningitis. NarE belongs to the ADP-ribosyltransferase family and catalyses the transfer of ADP-ribose moieties to arginine residues in target protein acceptors. Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and alter essential functions of eukaryotic cells. NarE was proposed to bind iron through a Fe-S center which is supposed to be implied in catalysis. We have produced and purified uniformly labeled 15 N-and 15 N/ 13 C-NarE and assigned backbone and side-chain resonances using multidimensional heteronuclear NMR spectroscopy. These assignments provide the starting point for the three-dimensional structure determination of NarE and the characterization of the role of the Fe-S center in the catalytic mechanism.
NhhA, Neisseria hia/hsf homologue, or GNA0992, is an oligomeric outer membrane protein of Neisser... more NhhA, Neisseria hia/hsf homologue, or GNA0992, is an oligomeric outer membrane protein of Neisseria meningitidis, recently included in the family of trimeric autotransporter adhesins. In this study we present the structural and functional characterization of this protein. By expressing in Escherichia coli the full-length gene, deletion mutants and chimeric proteins of NhhA, we demonstrated that the last 72 C-terminal residues are able to allow trimerization and localization of the N-terminal protein domain to the bacterial surface. In addition, we investigated on the possible role of NhhA in bacterial-host interaction events. We assessed in vitro the ability of recombinant purified NhhA to bind human epithelial cells as well as laminin and heparan sulphate. Furthermore, we shown that E. coli strain expressing NhhA was able to adhere to epithelial cells, and observed a reduced adherence in a meningococcal isogenic MC58DNhhA mutant. We concluded that this protein is a multifunctional adhesin, able to promote the bacterial adhesion to host cells and extracellular matrix components. Collectively, our results underline a putative role of NhhA in meningococcal pathogenesis and ascertain its structural and functional belonging to the emerging group of bacterial autotransporter adhesins with trimeric architecture.
The factor H binding protein (fHbp) is a 27-kDa membrane-anchored lipoprotein of Neisseria mening... more The factor H binding protein (fHbp) is a 27-kDa membrane-anchored lipoprotein of Neisseria meningitidis that allows the survival of the bacterium in human plasma; it is also a major component of a universal vaccine against meningococcus B.
Factor H-binding protein is a 27-kDa lipoprotein of Neisseria meningitidis discovered while scree... more Factor H-binding protein is a 27-kDa lipoprotein of Neisseria meningitidis discovered while screening the bacterial genome for vaccine candidates. In addition to being an important component of a vaccine against meningococcus in late stage of development, the protein is essential for pathogenesis because it allows the bacterium to survive and grow in human blood by binding the human complement factor H. We recently reported the solution structure of the C-terminal domain of factor H-binding protein, which contains the immunodominant epitopes. In the present study, we report the structure of the full-length molecule, determined by nuclear magnetic resonance spectroscopy. The protein is composed of two independent barrels connected by a short link. Mapping the residues recognized by monoclonal antibodies with bactericidal or factor H binding inhibition properties allowed us to predict the sites involved in the function of the protein. The structure therefore provides the basis for designing improved vaccine molecules.
International Journal of Medical Microbiology, 2004
With the advent of the genomic era, identification of bacterial factors involved in virulence is ... more With the advent of the genomic era, identification of bacterial factors involved in virulence is a different challenge. Given the vast amount of information available on toxins, in terms of sequence and 3D structure, and thanks to the growing number of sequenced bacterial genomes, it is possible to proceed by homology criteria to predict novel toxins in different microorganisms. ADP-ribosyltransferases constitute a class of functionally conserved enzymes, which display toxic activity in a variety of bacterial pathogens. Since these proteins play a key role in pathogenesis, they have been extensively characterized and successfully used as vaccine components and mucosal adjuvants. Therefore, the application of in silico analyses to identify novel members of this class of enzymes represents an important challenge in the genomic era. To address this subject, different groups have recently pursued homology-based procedures to screen bacterial genomes for novel, yet undiscovered ADP-ribosyltransferases (ADPRTs) and have identified more than twenty novel ADPRTs in Gram-positive and Gram-negative bacteria. We have developed a novel patternbased computational approach, which, flanked by secondary structure prediction tools, has allowed the identification of previously unrecognised putative ADPRTs. One of them, identified in Neisseria meningitidis has been extensively characterized and shown to possess the predicted enzymatic activity, suggesting a possible role of this protein in the virulence of Meningococcus.
Factor H-binding protein (fHBP; GNA1870) is one of the antigens of the recombinant vaccine agains... more Factor H-binding protein (fHBP; GNA1870) is one of the antigens of the recombinant vaccine against serogroup B Neisseria meningitidis, which has been developed using reverse vaccinology and is the basis of a meningococcal B vaccine entering phase III clinical trials. Binding of factor H (fH), an inhibitor of the complement alternative pathway, to fHBP enables N. meningitidis to evade killing by the innate immune system. All fHBP null mutant strains analyzed were sensitive to killing in ex vivo human whole blood and serum models of meningococcal bacteremia with respect to the isogenic wild-type strains. The fHBP mutant strains of MC58 and BZ83 (high fHBP expressors) survived in human blood and serum for less than 60 min (decrease of >2 log 10 CFU), while NZ98/254 (intermediate fHBP expressor) and 67/00 (low fHBP expressor) showed decreases of >1 log 10 CFU after 60 to 120 min of incubation. In addition, fHBP is important for survival in the presence of the antimicrobial peptide LL-37 (decrease of >3 log 10 CFU after 2 h of incubation), most likely due to electrostatic interactions between fHBP and the cationic LL-37 molecule. Hence, the expression of fHBP by N. meningitidis strains is important for survival in human blood and human serum and in the presence of LL-37, even at low levels. The functional significance of fHBP in mediating resistance to the human immune response, in addition to its widespread distribution and its ability to induce bactericidal antibodies, indicates that it is an important component of the serogroup B meningococcal vaccine.
The cDNA of the human muscle type acylphosphatase was isolated and characterized. The mRNA presen... more The cDNA of the human muscle type acylphosphatase was isolated and characterized. The mRNA presents a very long 5'-untranslated region, covering the first half of the molecule: 175 bases of this part were cloned and prediction of the possible secondary structure showed that a very stable stem-loop structure could be formed in that region. Moreover, an additional AUG triplet was found upstream of the start codon of the protein, defining an open reading frame of 60 codons which overlapped that of acylphosphatase. The possible regulatory effect on translation of this part of the mRNA molecule was studied by means of transient transfection experiments: a 10-fold decrease in the expression of a reporter protein and a dramatic decrease in the corresponding mRNA was observed, due to the presence of the 5'-untranslated region of acylphosphatase mRNA. Mutagenesis of the upstream AUG triplet eliminated mRNA instability, leading to the hypothesis that the product of the upstream open reading frame could play a role in this mechanism.
Previous experiments suggested that the upstream AUG triplet present in the 5P P untranslated reg... more Previous experiments suggested that the upstream AUG triplet present in the 5P P untranslated region (UTR) of muscle acylphosphatase mRNA is involved in the regulation of protein expression. In this paper, we study the involvement of the 5P PUTR secondary structure and upstream peptide on mRNA stability and protein translation. Our data, obtained using deletion and frame-shift mutants, demonstrate that the 5P PUTR controls protein expression regulating translation together with mRNA stability. Furthermore, we demonstrate that the inhibitory effect of the 5P PUTR of muscle acylphosphatase is relieved during the differentiation process in agreement with previous data reporting an increase of acylphosphatase content during cell differentiation. Finally, UV cross-linking experiments show that specific mRNA-binding proteins are associated with the 5P PUTR of the muscle acylphosphatase mRNA.
Common-type acylphosphatase is a small cytosolic enzyme whose catalytic properties and three-dime... more Common-type acylphosphatase is a small cytosolic enzyme whose catalytic properties and three-dimensional structure are known in detail. All the acidic residues of the enzyme have been replaced by noncharged residues in order to assess their contributions to the conformational stability of acylphosphatase. The enzymatic activity parameters and the conformational free energy of each mutant were determined by enzymatic activity assays and chemically induced unfolding, respectively. Some mutants exhibit very similar conformational stability, ⌬G(H 2 O), and specific activity values as compared to the wild-type enzyme. By contrast, six mutants show a significant reduction of conformational stability and two mutants are more stable than the wild-type protein. Although none of the mutated acidic residues is directly involved in the catalytic mechanism of the enzyme, our results indicate that mutations of residues located on the surface of the protein are responsible for a structural distortion which propagate up to the active site. We found a good correlation between the free energy of unfolding and the enzymatic activity of acylphosphatase. This suggests that enzymatic activity measurements can provide valuable indications on the conformational stability of acylphosphatase mutants, provided the mutated residue lies far apart from the active site. Moreover, our results indicate that the distortion of hydrogen bonds rather than the loss of electrostatic interactions, contributes to the decrease of the conformational stability of the protein.
Previous experiments suggested that the upstream AUG triplet present in the 5P P untranslated reg... more Previous experiments suggested that the upstream AUG triplet present in the 5P P untranslated region (UTR) of muscle acylphosphatase mRNA is involved in the regulation of protein expression. In this paper, we study the involvement of the 5P PUTR secondary structure and upstream peptide on mRNA stability and protein translation. Our data, obtained using deletion and frame-shift mutants, demonstrate that the 5P PUTR controls protein expression regulating translation together with mRNA stability. Furthermore, we demonstrate that the inhibitory effect of the 5P PUTR of muscle acylphosphatase is relieved during the differentiation process in agreement with previous data reporting an increase of acylphosphatase content during cell differentiation. Finally, UV cross-linking experiments show that specific mRNA-binding proteins are associated with the 5P PUTR of the muscle acylphosphatase mRNA.
The factor H binding protein (fHbp) is a 27-kDa membrane-anchored lipoprotein of Neisseria mening... more The factor H binding protein (fHbp) is a 27-kDa membrane-anchored lipoprotein of Neisseria meningitidis that allows the survival of the bacterium in human plasma; it is also a major component of a universal vaccine against meningococcus B.
International Journal of Medical Microbiology, 2004
With the advent of the genomic era, identification of bacterial factors involved in virulence is ... more With the advent of the genomic era, identification of bacterial factors involved in virulence is a different challenge. Given the vast amount of information available on toxins, in terms of sequence and 3D structure, and thanks to the growing number of sequenced bacterial genomes, it is possible to proceed by homology criteria to predict novel toxins in different microorganisms. ADP-ribosyltransferases constitute a class of functionally conserved enzymes, which display toxic activity in a variety of bacterial pathogens. Since these proteins play a key role in pathogenesis, they have been extensively characterized and successfully used as vaccine components and mucosal adjuvants. Therefore, the application of in silico analyses to identify novel members of this class of enzymes represents an important challenge in the genomic era. To address this subject, different groups have recently pursued homology-based procedures to screen bacterial genomes for novel, yet undiscovered ADP-ribosyltransferases (ADPRTs) and have identified more than twenty novel ADPRTs in Gram-positive and Gram-negative bacteria. We have developed a novel pattern-based computational approach, which, flanked by secondary structure prediction tools, has allowed the identification of previously unrecognised putative ADPRTs. One of them, identified in Neisseria meningitidis has been extensively characterized and shown to possess the predicted enzymatic activity, suggesting a possible role of this protein in the virulence of Meningococcus.
The knowledge of sequences and structures of proteins produced by microbial pathogens is continuo... more The knowledge of sequences and structures of proteins produced by microbial pathogens is continuously increasing. Besides offering the possibility to unravel the mechanisms of pathogenesis at molecular level, structural information provides new tools for vaccine development, such as the opportunity to improve viral and bacterial vaccine candidates by rational design. Structure-based rational design of antigens can optimize the epitope repertoire in terms of accessibility, stability and variability. In the present study, we used epitope mapping information on the well characterized antigen of Neisseria meningitidis factor H binding protein (fHbp) to engineer its gonococcal homologue Ghfp. Meningococcal fHbp is typically classified in three distinct antigenic variants. We introduced epitopes of fHbp variant 1 onto the surface of the Ghfp, which is naturally able to protect against meningococcal strains expressing fHbp of variants 2 and 3. Heterologous epitopes were successfully transplanted, as engineered Ghfp induced functional antibodies against all three fHbp variants. These results confirm that structural vaccinology represents a successful strategy to modulate immune responses as well as a powerful tool to investigate on extension and localization of immuno-dominant epitopes.
Mono ADP-ribosyltransferases (ADPRTs) are a class of functionally conserved enzymes present in pr... more Mono ADP-ribosyltransferases (ADPRTs) are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In bacteria, these enzymes often act as potent toxins and play an important role in pathogenesis. Here we report a profile-based computational approach that, assisted by secondary structure predictions, has allowed the identification of a previously undiscovered ADP-ribosyltransferase in Neisseria meningitidis (NarE). NarE shows structural homologies with E. coli heat-labile enterotoxin (LT) and cholera toxin (CT) and possesses ADP-ribosylating and NAD-glycohydrolase activities. As in the case of LT and CT, NarE catalyses the transfer of the ADP-ribose moiety to arginine residues. Despite the absence of a signal peptide, the protein is efficiently exported into the periplasm of Neisseria. The narE gene is present in 25 out of 43 strains analysed, is always present in ET-5 and Lineage 3 but absent in ET-37 and Cluster A4 hypervirulent lineages. When pre...
Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment... more Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution struc...
Common-type acylphosphatase is a small cytosolic enzyme whose catalytic properties and three-dime... more Common-type acylphosphatase is a small cytosolic enzyme whose catalytic properties and three-dimensional structure are known in detail. All the acidic residues of the enzyme have been replaced by noncharged residues in order to assess their contributions to the conformational stability of acylphosphatase. The enzymatic activity parameters and the conformational free energy of each mutant were determined by enzymatic activity assays
NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens r... more NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens responsible for meningitis. NarE belongs to the ADP-ribosyltransferase family and catalyses the transfer of ADP-ribose moieties to arginine residues in target protein acceptors. Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and alter essential functions of eukaryotic cells. NarE was proposed to bind iron through a Fe-S center which is supposed to be implied in catalysis. We have produced and purified uniformly labeled 15 N-and 15 N/ 13 C-NarE and assigned backbone and side-chain resonances using multidimensional heteronuclear NMR spectroscopy. These assignments provide the starting point for the three-dimensional structure determination of NarE and the characterization of the role of the Fe-S center in the catalytic mechanism.
NhhA, Neisseria hia/hsf homologue, or GNA0992, is an oligomeric outer membrane protein of Neisser... more NhhA, Neisseria hia/hsf homologue, or GNA0992, is an oligomeric outer membrane protein of Neisseria meningitidis, recently included in the family of trimeric autotransporter adhesins. In this study we present the structural and functional characterization of this protein. By expressing in Escherichia coli the full-length gene, deletion mutants and chimeric proteins of NhhA, we demonstrated that the last 72 C-terminal residues are able to allow trimerization and localization of the N-terminal protein domain to the bacterial surface. In addition, we investigated on the possible role of NhhA in bacterial-host interaction events. We assessed in vitro the ability of recombinant purified NhhA to bind human epithelial cells as well as laminin and heparan sulphate. Furthermore, we shown that E. coli strain expressing NhhA was able to adhere to epithelial cells, and observed a reduced adherence in a meningococcal isogenic MC58DNhhA mutant. We concluded that this protein is a multifunctional adhesin, able to promote the bacterial adhesion to host cells and extracellular matrix components. Collectively, our results underline a putative role of NhhA in meningococcal pathogenesis and ascertain its structural and functional belonging to the emerging group of bacterial autotransporter adhesins with trimeric architecture.
The factor H binding protein (fHbp) is a 27-kDa membrane-anchored lipoprotein of Neisseria mening... more The factor H binding protein (fHbp) is a 27-kDa membrane-anchored lipoprotein of Neisseria meningitidis that allows the survival of the bacterium in human plasma; it is also a major component of a universal vaccine against meningococcus B.
Factor H-binding protein is a 27-kDa lipoprotein of Neisseria meningitidis discovered while scree... more Factor H-binding protein is a 27-kDa lipoprotein of Neisseria meningitidis discovered while screening the bacterial genome for vaccine candidates. In addition to being an important component of a vaccine against meningococcus in late stage of development, the protein is essential for pathogenesis because it allows the bacterium to survive and grow in human blood by binding the human complement factor H. We recently reported the solution structure of the C-terminal domain of factor H-binding protein, which contains the immunodominant epitopes. In the present study, we report the structure of the full-length molecule, determined by nuclear magnetic resonance spectroscopy. The protein is composed of two independent barrels connected by a short link. Mapping the residues recognized by monoclonal antibodies with bactericidal or factor H binding inhibition properties allowed us to predict the sites involved in the function of the protein. The structure therefore provides the basis for designing improved vaccine molecules.
International Journal of Medical Microbiology, 2004
With the advent of the genomic era, identification of bacterial factors involved in virulence is ... more With the advent of the genomic era, identification of bacterial factors involved in virulence is a different challenge. Given the vast amount of information available on toxins, in terms of sequence and 3D structure, and thanks to the growing number of sequenced bacterial genomes, it is possible to proceed by homology criteria to predict novel toxins in different microorganisms. ADP-ribosyltransferases constitute a class of functionally conserved enzymes, which display toxic activity in a variety of bacterial pathogens. Since these proteins play a key role in pathogenesis, they have been extensively characterized and successfully used as vaccine components and mucosal adjuvants. Therefore, the application of in silico analyses to identify novel members of this class of enzymes represents an important challenge in the genomic era. To address this subject, different groups have recently pursued homology-based procedures to screen bacterial genomes for novel, yet undiscovered ADP-ribosyltransferases (ADPRTs) and have identified more than twenty novel ADPRTs in Gram-positive and Gram-negative bacteria. We have developed a novel patternbased computational approach, which, flanked by secondary structure prediction tools, has allowed the identification of previously unrecognised putative ADPRTs. One of them, identified in Neisseria meningitidis has been extensively characterized and shown to possess the predicted enzymatic activity, suggesting a possible role of this protein in the virulence of Meningococcus.
Factor H-binding protein (fHBP; GNA1870) is one of the antigens of the recombinant vaccine agains... more Factor H-binding protein (fHBP; GNA1870) is one of the antigens of the recombinant vaccine against serogroup B Neisseria meningitidis, which has been developed using reverse vaccinology and is the basis of a meningococcal B vaccine entering phase III clinical trials. Binding of factor H (fH), an inhibitor of the complement alternative pathway, to fHBP enables N. meningitidis to evade killing by the innate immune system. All fHBP null mutant strains analyzed were sensitive to killing in ex vivo human whole blood and serum models of meningococcal bacteremia with respect to the isogenic wild-type strains. The fHBP mutant strains of MC58 and BZ83 (high fHBP expressors) survived in human blood and serum for less than 60 min (decrease of >2 log 10 CFU), while NZ98/254 (intermediate fHBP expressor) and 67/00 (low fHBP expressor) showed decreases of >1 log 10 CFU after 60 to 120 min of incubation. In addition, fHBP is important for survival in the presence of the antimicrobial peptide LL-37 (decrease of >3 log 10 CFU after 2 h of incubation), most likely due to electrostatic interactions between fHBP and the cationic LL-37 molecule. Hence, the expression of fHBP by N. meningitidis strains is important for survival in human blood and human serum and in the presence of LL-37, even at low levels. The functional significance of fHBP in mediating resistance to the human immune response, in addition to its widespread distribution and its ability to induce bactericidal antibodies, indicates that it is an important component of the serogroup B meningococcal vaccine.
The cDNA of the human muscle type acylphosphatase was isolated and characterized. The mRNA presen... more The cDNA of the human muscle type acylphosphatase was isolated and characterized. The mRNA presents a very long 5'-untranslated region, covering the first half of the molecule: 175 bases of this part were cloned and prediction of the possible secondary structure showed that a very stable stem-loop structure could be formed in that region. Moreover, an additional AUG triplet was found upstream of the start codon of the protein, defining an open reading frame of 60 codons which overlapped that of acylphosphatase. The possible regulatory effect on translation of this part of the mRNA molecule was studied by means of transient transfection experiments: a 10-fold decrease in the expression of a reporter protein and a dramatic decrease in the corresponding mRNA was observed, due to the presence of the 5'-untranslated region of acylphosphatase mRNA. Mutagenesis of the upstream AUG triplet eliminated mRNA instability, leading to the hypothesis that the product of the upstream open reading frame could play a role in this mechanism.
Previous experiments suggested that the upstream AUG triplet present in the 5P P untranslated reg... more Previous experiments suggested that the upstream AUG triplet present in the 5P P untranslated region (UTR) of muscle acylphosphatase mRNA is involved in the regulation of protein expression. In this paper, we study the involvement of the 5P PUTR secondary structure and upstream peptide on mRNA stability and protein translation. Our data, obtained using deletion and frame-shift mutants, demonstrate that the 5P PUTR controls protein expression regulating translation together with mRNA stability. Furthermore, we demonstrate that the inhibitory effect of the 5P PUTR of muscle acylphosphatase is relieved during the differentiation process in agreement with previous data reporting an increase of acylphosphatase content during cell differentiation. Finally, UV cross-linking experiments show that specific mRNA-binding proteins are associated with the 5P PUTR of the muscle acylphosphatase mRNA.
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Papers by Daniele Veggi