| Abstract: | Thirty percent of Streptococcus pneumoniae isolates contain pilus islet 1, coding for a pilus composed of the backbone subunit RrgB and two ancillary proteins, RrgA and RrgC. RrgA is the major determinant of in vitro adhesion associated with pilus 1, is protective in vivo in mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the “head” of the protein, which contains the putative binding domains, whereas the elongated “stalk” was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the “head” domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.Streptococcus pneumoniae is a main determinant of respiratory tract infections, such as otitis media, sinusitis, and community-acquired pneumonia, and is also responsible for invasive diseases, such as bacteremic pneumonia and meningitis (16, 17, 30, 43, 46, 50, 55). Nonetheless, pneumococci are normal components of the human commensal flora, asymptomatically colonizing the upper respiratory tracts of both children and healthy adults. Colonization is commonly followed by horizontal transmission of S. pneumoniae, leading to its spread within the community (4, 19, 33). Current glycoconjugate vaccines are efficacious against invasive disease caused by serotypes included in the vaccines; however, their potential to prevent carriage and related mucosal diseases, such as otitis media, is not optimal (10, 12, 23, 29, 34, 48). Furthermore, the partial geographic coverage and the phenomenon of serotype replacement associated with the introduction of the 7-valent conjugate vaccine limit to some extent its long-term effectiveness (11, 20, 31, 37). For these reasons, current research is focused on the identification of protein vaccine candidates able to elicit serotype-independent protection against S. pneumoniae infection. In this context, colonization could represent a critical point of intervention, and bacterial components involved in these mechanisms should be studied in order to determine their value as vaccine candidates.Adhesion of bacteria to the mucosa is considered an essential early step in the colonization process. The ability of S. pneumoniae to adhere to epithelial cells has been ascribed to a number of surface-exposed proteins, including PspC, PsaA, PsrP, PfbB, NanA, PavA, and pili (3, 21, 41, 42, 45, 49, 54).Pili were recently discovered in many Gram-positive pathogens, and although their biological function has not been fully elucidated, their presence has been mostly related to bacterial adhesion, biofilm formation, and translocation of epithelial barriers (1, 13, 35, 47). These structures are composed of subunits covalently linked by means of intermolecular isopeptide bonds (32, 36, 51-53). Furthermore, intramolecular isopeptide bonds have been found in most pilus subunits characterized to date (9, 26-28). These bonds may play a critical role in maintaining pilus integrity in the face of severe mechanical and chemical stress while bound to host cells and thus may provide a functional mode of stabilization for cell surface proteins involved in host pathogenesis.In S. pneumoniae, pili (pilus 1 and pilus 2) are encoded by two genomic islets (pilus islet 1 [PI-1] and PI-2) that are not present in all pneumococcal clinical isolates. A number of molecular epidemiological studies have highlighted the presence of PI-1 as a clonal property of S. pneumoniae isolates and have defined, based on sequence analysis, the classification of PI-1 into three major clades (2, 5, 7, 25, 38, 39). Mutants lacking PI-1 are impaired in adhesion to cultured epithelial cells in vitro and are less virulent in murine models of colonization, pneumonia, and bacteremia (6, 41). Interestingly, pilus 1 expression is known to increase host inflammatory responses that might disrupt the mucosal barrier and facilitate subsequent invasion by the bacteria (6).Pneumococcal pilus 1 is composed of three subunits (RrgA, RrgB, and RrgC); RrgB is the backbone component, and RrgA is the major ancillary protein, localized at the pilus tip and responsible for the adhesion properties of the pilus, whereas RrgC is the minor ancillary protein, likely located at the pilus base (21, 22, 41). In terms of sequence variability, RrgB is classified into three variants and RrgC is conserved, whereas RrgA exists in two major variants (clades I and II) (38). The recombinant form of RrgA clade I adheres in vitro to cultured A549 lung epithelial cells, as well as to purified extracellular matrix (ECM) components (collagen I, fibronectin, and laminin) (21, 41). In addition, RrgA, along with the other two pilus 1 components, is able to elicit protection from lethal challenge with the homologous strain in mouse models of active and passive immunization (18).In this work, we investigated whether the differences between the two variants had an effect on the biochemical characteristics, biological function, and immunological properties of the molecule. We found that (i) sequence variability was restricted to the “head” domain of RrgA, containing the putative adhesive motifs; (ii) the two RrgA variants were resistant to proteolytic cleavage, and this feature was dependent on the presence of intramolecular isopeptide bonds; (iii) the two variants were able to adhere to epithelial cells and ECM components at comparable levels, whereas a mutant (Asp444Ala) in the RGD tripeptide showed reduced binding; (iv) none of the individual fragments encompassing the N-terminal (NT), central (CP), and C-terminal (CT) portions of RrgA was able to maintain adhesive capacity; (v) antibodies against these fragments revealed the N terminus to be less accessible than the remaining portion of the molecule on the native pilus; and, finally, (vi) antibodies raised against each of the two RrgA variants were cross-reactive and cross-protective in murine passive-immunization studies. |
