Understanding the molecular determinants driving the immunological specificity of the protective pilus 2a backbone protein of Group B Streptococcus

The pilus 2a backbone protein (BP-2a) is one of the most structurally and functionally characterized components of a potential vaccine formulation against Group B Streptococcus. It is characterized by six main immunologically distinct allelic variants, each inducing variant-specific protection. To investigate the molecular determinants driving the variant immunogenic specificity of BP-2a, in terms of single residue contributions, we generated six monoclonal antibodies against a specific protein variant based on their capability to recognize the polymerized pili structure on the bacterial surface. Three mAbs were also able to induce complement-dependent opsonophagocytosis killing of live GBS and target the same linear epitope present in the structurally defined and immunodominant domain D3 of the protein. Molecular docking between the modelled scFv antibody sequences and the BP-2a crystal structure revealed the potential role at the binding interface of some non-conserved antigen residues. Mutagenesis analysis confirmed the necessity of a perfect balance between charges, size and polarity at the binding interface to obtain specific binding of mAbs to the protein antigen for a neutralizing response

A direct role for SNX9 in the biogenesis of filopodia.

Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia

Sortase A Substrate Specificity in GBS Pilus 2a Cell Wall Anchoring

Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b), whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA), whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2) is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtAΔN40) able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a) and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtAΔN40 does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein). Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus

A direct role for SNX9 in the biogenesis of filopodia.

Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia

Understanding the molecular determinants driving the immunological specificity of the protective pilus 2a backbone protein of Group B Streptococcus

The pilus 2a backbone protein (BP-2a) is one of the most structurally and functionally characterized components of a potential vaccine formulation against Group B Streptococcus. It is characterized by six main immunologically distinct allelic variants, each inducing variant-specific protection. To investigate the molecular determinants driving the variant immunogenic specificity of BP-2a, in terms of single residue contributions, we generated six monoclonal antibodies against a specific protein variant based on their capability to recognize the polymerized pili structure on the bacterial surface. Three mAbs were also able to induce complement-dependent opsonophagocytosis killing of live GBS and target the same linear epitope present in the structurally defined and immunodominant domain D3 of the protein. Molecular docking between the modelled scFv antibody sequences and the BP-2a crystal structure revealed the potential role at the binding interface of some non-conserved antigen residues. Mutagenesis analysis confirmed the necessity of a perfect balance between charges, size and polarity at the binding interface to obtain specific binding of mAbs to the protein antigen for a neutralizing response

Structure and Assembly of Group B Streptococcus Pilus 2b Backbone Protein

<div><p>Group B <i>Streptococcus</i> (GBS) is a major cause of invasive disease in infants. Like other Gram-positive bacteria, GBS uses a sortase C-catalyzed transpeptidation mechanism to generate cell surface pili from backbone and ancillary pilin precursor substrates. The three pilus types identified in GBS contain structural subunits that are highly immunogenic and are promising candidates for the development of a broadly-protective vaccine. Here we report the X-ray crystal structure of the backbone protein of pilus 2b (BP-2b) at 1.06Å resolution. The structure reveals a classical IgG-like fold typical of the pilin subunits of other Gram-positive bacteria. The crystallized portion of the protein (residues 185-468) encompasses domains D2 and D3 that together confer high stability to the protein due to the presence of an internal isopeptide bond within each domain. The D2+D3 region, lacking the N-terminal D1 domain, was as potent as the entire protein in conferring protection against GBS challenge in a well-established mouse model. By site-directed mutagenesis and complementation studies in GBS knock-out strains we identified the residues and motives essential for assembly of the BP-2b monomers into high-molecular weight complexes, thus providing new insights into pilus 2b polymerization.</p></div

Pili genes pattern in Group B streptococci from newborn infections and pregnant women in Europe (DEVANI Project)

Objectives Evaluation of the presence and expression of genes coding for pili in a collection of group B streptococcci (GBS) isolated from newborn infection and pregnant women in the course of the DEVANI (Design of a Vaccine Against Neonatal Infection) project. Methods GBS isolates from pregnant women (PW) and cases of newborn infection (NI) were collected in 8 European countries (Belgium, Bulgaria, Czech Republic, Denmark, Germany, Italy, Spain, United Kingdom) during 2009/10 under the auspices of DEVANI. Total no. of strains examined was 1078 and 192 from PW and NI, respectively. Isolates were screened by multiplex PCR and FACS analysis to evaluate respectively gene presence and surface-exposure of pili. Results The most common gene patterns found were PI-2a alone, PI 1+2a and PI 1+2b, while the PI-2b gene alone was very rare. The most prominent result was that a majority of isolates from NI carried the PI-1+2b gene pattern, while the most common pattern among PW was PI-1+2a. Most of analyzed strains express at least one pilus on their surface. Conclusions All isolates contained at least one gene coding for pili. When present pili 2a and 2b were highly surface exposed

Biochemical characterization of different BP-2b constructs.

<p><b>(A)</b> Time course of the trypsin-proteolysis reactions at 37°C of BP-2b full length and fragments, analyzed by SDS-PAGE. Different digestion patterns can be observed for the different constructs. Asterisks indicate the not-digested proteins. (<b>B)</b> Differential Scanning Fluorimetry (DSF) analysis of BP-2b proteins (D1+D2+D3, D2+D3 and single domains D1, D2, D3) in presence of Sypro orange showed different thermal stabilities. Graph shows the fluorescence intensity <i>vs</i>. the temperature for the unfolding different BP-2b constructs. (<b>C)</b> Correlation of BP-2b melting temperature with the concentration of Ca²⁺.</p

Structural comparisons of BP-2b_D2+D3 with other pilin backbone proteins.

<p>(<b>A)</b> BP-2b (blue cartoon) is shown overlaid onto: the pilus backbone protein RrgB (pdb 2x9x, red cartoon, left), the major pilin protein GBS80 (pdb 3pf2, green cartoon, middle), and on the major pilin protein BP-2a (pdb 2xtl, pink cartoon, right). (<b>B)</b> Domain architecture of GBS backbone proteins from pilus 1 (BP-1), pilus 2a (BP-2a) and pilus 2b (BP-2b). The proteins are comprised of a signal peptide (SP) at the N-terminus and a C-terminal LPXTG-like motif (in red) linked to the transmembrane domain (TM). BP-1 and BP-2b contain three domains, while BP-2a four domains. The pilin motif involved in pilus polymerization is located near the D1–D2 domain linker while the E-box is located close to the sorting signal. Residues involved in isopeptide bonds are indicated by black bars. Domains present in the crystal structures are included into the box outlined with dashed lines.</p

Multiple structural alignment of BP-2b protein with other known structures using the DALI server.

<p>Hits are ranked by Z-Score with best hits at the top of the table.</p><p><i>PDB</i>: Protein Data Bank</p><p><i>rmsd</i>: root-mean-square deviation of Cα atoms of superimposed proteins in Angstroms</p><p><i>lali</i>: number of structurally equivalent positions</p><p><i>nres</i>: number of structurally equivalent aligned residues</p><p><i>%ide</i>: percentage of amino acid identity in aligned positions</p><p>Multiple structural alignment of BP-2b protein with other known structures using the DALI server.</p