Structural and functional characterization of Group B Streptococcus pilus 2b

Group B Streptococcus (GBS) is a Gram-positive human pathogen representing one of the most common causes of life-threatening bacterial infections such as sepsis and meningitis in neonates. Covalently polymerized pilus-like structures have been discovered in GBS as important virulence factors as well as vaccine candidates. Pili are protein polymers forming long and thin filamentous structures protruding from bacterial cells, mediating adhesion and colonization to host cells. Gram-positive bacteria, including GBS, build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates that are the backbone protein forming the pilus shaft and two ancillary proteins. Also the cell-wall anchoring of the pilus polymers made of covalently linked pilin subunits is mediated by a sortase enzyme. GBS expresses three structurally distinct pilus types (type 1, 2a and 2b). Although the mechanisms of assembly and cell wall anchoring of GBS types 1 and 2a pili have been investigated, those of pilus 2b are not understood until now. Pilus 2b is frequently found in ST-17 strains that are mostly associated with meningitis and high mortality rate especially in infants. In this work the assembly mechanism of GBS pilus type 2b has been elucidated by dissecting through genetic, biochemical and structural studies the role of the two pilus-associated sortases. The most significant findings show that pilus 2b assembly appears “non-canonical”, differing significantly from current pilus assembly models in Gram-positive pathogens. Only sortase-C1 is involved in pilin polymerization, while the sortase-C2 does not act as a pilin polymerase, but it is involved in cell-wall pilus anchoring. Our findings provide new insights into pili biogenesis in Gram-positive bacteria. Moreover, the role of this pilus type during host infection has been investigated. By using a mouse model of meningitis we demonstrated that type 2b pilus contributes to pathogenesis of meningitis in vivo.

Polymerizing activity and regulation of group B Streptococcus pilus 2a sortase C1

Group B Streptococcus [GBS; Streptococcus agalactiae] is the leading cause of life-threatening diseases in newborn and is also becoming a common cause of invasive diseases in non-pregnant, elderly and immune-compromised adults. Pili, long filamentous fibers protruding from the bacterial surface, have been discovered in GBS, as important virulence factors and vaccine candidates. Gram-positive bacteria build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates. Despite the availability of several crystal structures, pilus-related C sortases remain poorly characterized to date and their mechanisms of transpeptidation and regulation need to be further investigated. The available three-dimensional structures of these enzymes reveal a typical sortase fold except for the presence of a unique feature represented by an N-terminal highly flexible loop, known as the “lid”. This region interacts with the residues composing the catalytic triad and covers the active site, thus maintaining the enzyme in an auto-inhibited state and preventing the accessibility to the substrate. It is believed that enzyme activation may occur only after lid displacement from the catalytic domain. In this work we provide the first direct evidence of the regulatory role of the lid, demonstrating that it is possible to obtain in vitro an efficient polymerization of pilin subunits using an active C sortase lid mutant carrying a single residue mutation in the lid region. Moreover, biochemical analyses of this recombinant mutant reveal that the lid confers thermodynamic and proteolytic stability to the enzyme. A further characterization of this sortase active mutant showed promiscuity in the substrate recognition, as it is able to polymerize different LPXTG-proteins in vitro.

Studies of OPA1 pathogenic mechanisms in Dominant Optic Atrophy and novel protein function in mitochondrial DNA stability

The mitochondrion is an essential cytoplasmic organelle that provides most of the energy necessary for eukaryotic cell physiology. Mitochondrial structure and functions are maintained by proteins of both mitochondrial and nuclear origin. These organelles are organized in an extended network that dynamically fuses and divides. Mitochondrial morphology results from the equilibrium between fusion and fission processes, controlled by a family of “mitochondria-shaping” proteins. It is becoming clear that defects in mitochondrial dynamics can impair mitochondrial respiration, morphology and motility, leading to apoptotic cell death in vitro and more or less severe neurodegenerative disorders in vivo in humans. Mutations in OPA1, a nuclear encoded mitochondrial protein, cause autosomal Dominant Optic Atrophy (DOA), a heterogeneous blinding disease characterized by retinal ganglion cell degeneration leading to optic neuropathy (Delettre et al., 2000; Alexander et al., 2000). OPA1 is a mitochondrial dynamin-related guanosine triphosphatase (GTPase) protein involved in mitochondrial network dynamics, cytochrome c storage and apoptosis. This protein is anchored or associated on the inner mitochondrial membrane facing the intermembrane space. Eight OPA1 isoforms resulting from alternative splicing combinations of exon 4, 4b and 5b have been described (Delettre et al., 2001). These variants greatly vary among diverse organs and the presence of specific isoforms has been associated with various mitochondrial functions. The different spliced exons encode domains included in the amino-terminal region and contribute to determine OPA1 functions (Olichon et al., 2006). It has been shown that exon 4, that is conserved throughout evolution, confers functions to OPA1 involved in maintenance of the mitochondrial membrane potential and in the fusion of the network. Conversely, exon 4b and exon 5b, which are vertebrate specific, are involved in regulation of cytochrome c release from mitochondria, and activation of apoptosis, a process restricted to vertebrates (Olichon et al., 2007). While Mgm1p has been identified thanks to its role in mtDNA maintenance, it is only recently that OPA1 has been linked to mtDNA stability. Missense mutations in OPA1 cause accumulation of multiple deletions in skeletal muscle. The syndrome associated to these mutations (DOA-1 plus) is complex, consisting of a combination of dominant optic atrophy, progressive external ophtalmoplegia, peripheral neuropathy, ataxia and deafness (Amati- Bonneau et al., 2008; Hudson et al., 2008). OPA1 is the fifth gene associated with mtDNA “breakage syndrome” together with ANT1, PolG1-2 and TYMP (Spinazzola et al., 2009). In this thesis we show for the first time that specific OPA1 isoforms associated to exon 4b are important for mtDNA stability, by anchoring the nucleoids to the inner mitochondrial membrane. Our results clearly demonstrate that OPA1 isoforms including exon 4b are intimately associated to the maintenance of the mitochondrial genome, as their silencing leads to mtDNA depletion. The mechanism leading to mtDNA loss is associated with replication inhibition in cells where exon 4b containing isoforms were down-regulated. Furthermore silencing of exon 4b associated isoforms is responsible for alteration in mtDNA-nucleoids distribution in the mitochondrial network. In this study it was evidenced that OPA1 exon 4b isoform is cleaved to provide a 10kd peptide embedded in the inner membrane by a second transmembrane domain, that seems to be crucial for mitochondrial genome maintenance and does correspond to the second transmembrane domain of the yeasts orthologue encoded by MGM1 or Msp1, which is also mandatory for this process (Diot et al., 2009; Herlan et al., 2003). Furthermore in this thesis we show that the NT-OPA1-exon 4b peptide co-immuno-precipitates with mtDNA and specifically interacts with two major components of the mitochondrial nucleoids: the polymerase gamma and Tfam. Thus, from these experiments the conclusion is that NT-OPA1- exon 4b peptide contributes to the nucleoid anchoring in the inner mitochondrial membrane, a process that is required for the initiation of mtDNA replication and for the distribution of nucleoids along the network. These data provide new crucial insights in understanding the mechanism involved in maintenance of mtDNA integrity, because they clearly demonstrate that, besides genes implicated in mtDNA replications (i.e. polymerase gamma, Tfam, twinkle and genes involved in the nucleotide pool metabolism), OPA1 and mitochondrial membrane dynamics play also an important role. Noticeably, the effect on mtDNA is different depending on the specific OPA1 isoforms down-regulated, suggesting the involvement of two different combined mechanisms. Over two hundred OPA1 mutations, spread throughout the coding region of the gene, have been described to date, including substitutions, deletions or insertions. Some mutations are predicted to generate a truncated protein inducing haploinsufficiency, whereas the missense nucleotide substitutions result in aminoacidic changes which affect conserved positions of the OPA1 protein. So far, the functional consequences of OPA1 mutations in cells from DOA patients are poorly understood. Phosphorus MR spectroscopy in patients with the c.2708delTTAG deletion revealed a defect in oxidative phosphorylation in muscles (Lodi et al., 2004). An energetic impairment has been also show in fibroblasts with the severe OPA1 R445H mutation (Amati-Bonneau et al., 2005). It has been previously reported by our group that OPA1 mutations leading to haploinsufficiency are associated in fibroblasts to an oxidative phosphorylation dysfunction, mainly involving the respiratory complex I (Zanna et al., 2008). In this study we have evaluated the energetic efficiency of a panel of skin fibroblasts derived from DOA patients, five fibroblast cell lines with OPA1 mutations causing haploinsufficiency (DOA-H) and two cell lines bearing mis-sense aminoacidic substitutions (DOA-AA), and compared with control fibroblasts. Although both types of DOA fibroblasts maintained a similar ATP content when incubated in a glucose-free medium, i.e. when forced to utilize the oxidative phosphorylation only to produce ATP, the mitochondrial ATP synthesis through complex I, measured in digitonin-permeabilized cells, was significantly reduced in cells with OPA1 haploinsufficiency only, whereas it was similar to controls in cells with the missense substitutions. Furthermore, evaluation of the mitochondrial membrane potential (DYm) in the two fibroblast lines DOA-AA and in two DOA-H fibroblasts, namely those bearing the c.2819-2A>C mutation and the c.2708delTTAG microdeletion, revealed an anomalous depolarizing response to oligomycin in DOA-H cell lines only. This finding clearly supports the hypothesis that these mutations cause a significant alteration in the respiratory chain function, which can be unmasked only when the operation of the ATP synthase is prevented. Noticeably, oligomycin-induced depolarization in these cells was almost completely prevented by preincubation with cyclosporin A, a well known inhibitor of the permeability transition pore (PTP). This results is very important because it suggests for the first time that the voltage threshold for PTP opening is altered in DOA-H fibroblasts. Although this issue has not yet been addressed in the present study, several are the mechanisms that have been proposed to lead to PTP deregulation, including in particular increased reactive oxygen species production and alteration of Ca2+ homeostasis, whose role in DOA fibroblasts PTP opening is currently under investigation. Identification of the mechanisms leading to altered threshold for PTP regulation will help our understanding of the pathophysiology of DOA, but also provide a strategy for therapeutic intervention.