Characterization studies of Streptococcus agalactiae pilus type 2b and Srt2-2b sortase enzyme

Group B Streptococcus or GBS (also referred as Streptococcus agalactiae) 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 and infants. Covalently polymerized pilus-like structures have been discovered in GBS as important virulence factors as well as vaccine candidates. Pili are protein polymers that form long and thin filamentous structures protruding from bacterial cells, mediating adhesion and colonization to host cells and other activities involved in the virulence of the bacterium. 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 (BP) forming the pilus shaft and ancillary proteins (APs). GBS expresses three structurally distinct pilus types (type 1, 2a and 2b). The biogenesis of the pilus type 2b in terms of protein polymerization and cell-wall anchoring of the polymerized pilus structure has been recently investigated in our laboratory and revealed significant differences from the current model of pilus assembly in Gram-positive pathogens. Genetic and biochemical analyses showed that only one sortase (SrtC1-2b) is responsible for protein polymerization, whereas the second one (Srt2-2b) does not act as a pilin polymerase and is instead involved in pilus anchoring to the cell-wall by targeting the minor ancillary pilin subunit. Pilus 2b is frequently found in ST-17 serotype III strains mostly associated with meningitis and high mortality rate especially in infants. However, this pilus type remains the less characterized among GBS pili. In this work, we first present a systematic review and meta-analysis of the distribution of pilus islands in GBS, with a special focus on pilus 2b prevalence; this analysis was conducted on a large panel of isolates associated with colonization, neonatal invasive infections and adult disease, derived from different epidemiological studies and collected by our laboratory during the last decade. We also present the results of a genomic analysis of pilus 2b gene conservation that we have conducted starting from a dataset of 140 complete genomes available in the public databases. Finally, the role of Srt2-2b sortase has been investigated in detail by genomic, biochemical and structural studies in order to fully understand the molecular basis of the mechanism of pilus 2b biogenesis. The Results section in this Thesis report is preceded by an Introduction summarizing the state of the art on GBS molecular pathogenesis, GBS pili, and available biochemical/structural data on the role of sortase enzymes in Gram-positive pilus assembly and cell-wall anchoring. The meta-analysis of pilus distribution confirmed that pilus 2b was strongly associated to serotype III strains belonging to the hypervirulent ST-17 lineage and was the most represented among Late Onset Disease (LOD) strains. Moreover, DNA sequencing analysis showed that pilus 2b proteins are well conserved among GBS strains. Sequence and phylogenetic analysis of pilus 2b sortases in comparison with other Gram-positive sortases from different classes clearly indicated that GBS Srt2-2b is not a canonical C sortase and does not belong to any of the known sortase families. Indeed, Srt2-2b presents a shorter aminoacid sequence and completely lacks the canonical N-terminal regulatory “lid” containing the DPF\Y\W motif and the C-terminal transmembrane (TM) helix. Moreover, Srt2-b contains two cysteine residues (C115 and C192) in addition to the one present in the catalytic triad (H117, C180 and R187). Site-specific mutagenesis and biochemical assays confirmed the essential function of the catalytic triad (H117, C180 and R187) and provided evidence on the role of C115 and C192 in the regulation of Srt2-2b activity via a redox mechanism, alternative to the lid displacing mechanism typical of pilin-polymerizing class C sortases. A complete 3D structure of Srt2-2b protein could elucidate the specific role of Srt2-2b and provide new insights in pilus 2b biogenesis. Unfortunately, only a partial backbone assignment has been obtained so far during the Nuclear Magnetic Resonance (NMR) studies we have conducted for structural determination of Srt2-2b. These results indicate a high mobility of the enzyme and internal disorder and are consistent with the failing in obtaining crystals for Srt2-2b.