In addition, one or more accessory subunits are incorporated into the pilus by an as yet unknown mechanism that requires the pilusspecific sortase. The crystallographic structures of two major Nap-FF pilins have now shown that the E-box domain is involved in the formation of intramolecular isopeptide bond conferring higher stability to the pilin monomer. Then, during the final step, the pilus fiber is covalently linked to the peptidoglycan by either the pilus-specific or the housekeeping sortase. This mechanism of pilus assembly catalyzed by class C sortases has now been characterized in several gram-positive pathogens using similar genetic and biochemical analyses. Three genomic loci have been described in GBS strains, the latter two being mutually exclusive as they are located at the same chromosomal location. In a survey of 289 GBS clinical isolates, PI-1, PI-2a, and PI-2b were detected in 72%, 73%, and 27% of the strains, the combination of PI-1 + PI-2a being the most frequent. We and others previously carried out a detailed structural and functional analysis of the pilus locus PI-2a in GBS strain NEM316. This locus encodes the three structural pilus subunits PilA, PilB, and PilC whose assembly involves two class C sortases. PilB, the bona fide pilin, is the major component; PilC is a minor associated component mainly localized at the base of the pilus ; and PilA is the adhesin located at intervals along the pilus backbone. The PI-2a GBS pili have also been implicated in mediating attachment to human epithelial cells, in biofilm formation, in the adhesion and invasion of brain microvascular endothelial cells, and in promoting transepithelial migration. Intriguingly, the pilin subunit PilB of PI-2a was also reported to mediate resistance to cathelicidin antimicrobial peptide and SPP86 phagocyte killing, to increase bloodstream survival, and to confer virulence in a mouse challenge model. Here, we re-investigate the contribution of PilB in the virulence of strain NEM316 using two different mice models and in resistance to innate host immune defenses by testing GBS survival to killing by macrophages or antimicrobial peptides.