SummaryWe have recently reported the presence of covalently linked pilus-like structures in the human pathogen, Group B Streptococcus (GBS). The pilus operon codes for three proteins which contain the conserved amino acid motif, LPXTG, associated with cell wall-anchored proteins together with two genes coding for sortase enzymes. Analysis of the eight sequenced genomes of GBS has led to the identification of a second, related genomic island of which there are two variants, each containing genes coding for proteins with LPXTG motifs and sortases. Here we show that both variant islands also code for pilus-like structures. Furthermore, we provide a thorough description and characterization of the genomic organization of the islands and the role of each protein in the assembly of the pili. For each pilus, polymerization of one of the three component proteins is essential for incorporation of the other two proteins into the pilus structure. In addition, two sortases are required for complete pilus assembly, each with specificity for one of the pilus components. A component protein of one of the newly identified pili is also a previously identified protective antigen and a second component of this pilus is shown to confer protection against GBS challenge. We propose that pilus-like structures are important virulence factors and potential vaccine candidates.

Pili are essential virulence factors in many Gram-negative bacteria; however, they have not been described in most important Gram-positive pathogens. While screening the sequence of multiple genomes of Group B Streptococcus , we identified protective antigens that formed high molecular weight polymers. Immunogold electron microscopy revealed that the structures have a pilus-like form. These large structures have gone unrecognized in decades of studies of Group B Streptococcus .

Significance Several human pathogens exploit genomic variability to adapt to the host environment. Genome sequencing of collections of isolates and classification of strains according to their genomic content are pivotal to the formulation of vaccines able to elicit broad protection. We sequenced a collection of carriage and disease isolates of nontypeable Haemophilus influenzae , a component of the microbial flora of the upper respiratory tract that can cause a spectrum of diseases, including otitis media and meningitis. We identified distinct evolutionary clades that correlate with the presence of selected surface-associated proteins and virulence determinants. The high-resolution definition of the population structure of nontypeable Haemophilus influenzae allowed by whole-genome sequencing will be key for the development of efficacious containment strategies for this important human pathogen.

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