High-Affinity Interaction between Fibronectin and the Group B Streptococcal C5a Peptidase Is Unaffected by a Naturally Occurring Four-Amino-Acid Deletion That Eliminates Peptidase Activity

Abstract

The streptococcal C5a peptidase (ScpB) of group B streptococci (GBS) is found in virtually all clinical GBS isolates and is required for mucosal colonization in a neonatal mouse model. ScpB inhibits neutrophil chemotaxis by enzymatically cleaving the complement component C5a. We previously identified a second function of ScpB as a fibronectin (Fn) adhesin using phage display. However, phage display can identify low-affinity interactions. We therefore measured the affinity of both full-length recombinant ScpB (FL-ScpB) and the 110-amino-acid phage display fragment (Scp-PDF) for immobilized Fn using surface plasmon resonance. The affinity for Fn was very high for both FL-ScpB (equilibrium dissociation constant [K D ] ‫؍‬ 4.0 nM) and Scp-PDF (K D ‫؍‬ 4.4 nM) and is consistent with a biologically significant role for the adhesin activity of ScpB. We also studied the Fn adhesin activity of a common natural variant of ScpB (ScpB⌬) that contains a 4-amino-acid deletion that eliminates peptidase activity. The integrity of scpB is otherwise maintained, suggesting that the Fn adhesin activity of ScpB may be responsible for its conservation in these strains. The affinities of both FL-ScpB⌬ (K D ‫؍‬ 2.4 nM) and ScpB⌬-PDF (K D ‫؍‬ 1.4 nM) for Fn are unaffected by the deletion. Complementation in trans by both scpB and scpB⌬ corrected the Fn-binding defect of an scpB deletion mutant GBS strain to an identical degree. The high affinity of ScpB for Fn and the maintenance of this affinity in ScpB⌬ support our hypothesis that the Fn adhesin activity of scpB plays a role in virulence.Group B streptococci (GBS) are a leading cause of sepsis and meningitis in newborns (2) and an emerging cause of serious bacterial infections in immunocompromised adults and the elderly (12). Recent advances in prevention have significantly reduced the burden of early-onset neonatal disease (onset at Ͻ7 days of age) but have not had an impact on the incidence of late-onset disease (onset at 7 to 90 days of age) (25).Evasion of host defenses is a key component of bacterial virulence. One major host defense is complement, which is involved in a variety of functions including opsonization and chemotaxis. Complement activation results in the proteolytic cleavage of C5 to generate C5a. C5a is a potent chemotactic factor that plays an important role in recruiting neutrophils to sites of inflammation. The streptococcal C5a peptidase (ScpB) is a cell surface endopeptidase produced by GBS that inactivates C5a (9). The role of scpB in pulmonary disease by GBS was recently investigated (7). Isogenic scpB mutant GBS strains showed a 5-log decrease in their ability to colonize the lungs of adult mice, demonstrating that scpB plays a significant role in mucosal colonization. These results suggested that the ability to cleave C5a is required for mucosal colonization.However, epidemiologic studies of clinical isolates of GBS called into question the role of the peptidase activity of ScpB in virulence. The scpB gene is conserved among clinical isolates, suggesting tha...

StreptococcusAdherence and Colonization

“…In addition, C5a peptidases of S. pyogenes (ScpA) and S. agalactiae (ScpB) (Fig. 5) are able to bind Fn with high affinity (96,595), but this is not associated with the protease cleavage site or with Fn-binding repeats (the repeats shown in Fig. 5 are Fn type III domains).…”

Human Serum Induces Streptococcal C5a Peptidase Expression

“…Interestingly, all human but only some bovine S. agalactiae isolates possess the scpB gene (14,17). Genetic polymorphisms alter the functional activity of C5a peptidase (5) but do not affect its ability to bind fibronectin (40). Overall, its conserved nucleotide sequence in several ␤-hemolytic streptococcal species, its ubiquitous expression, and its surface localization make it a good vaccine candidate for S. agalactiae, as well as S. pyogenes, infections (10,21,29,32).…”

Interactions of the streptococcal C5a peptidase with human fibronectin

“…If the ScpB-Fn binding depends only on whether Fn is adsorbed or in solution, then the binding force should not vary with surface concentration, and the frequency of interaction should increase or decrease depending on whether or the ScpB binding site is hidden or exposed by the structural changes of Fn film. The force distribution should be bimodal for all surface concentrations since there are two interaction sites for the Scp/Fn system [22]. If the ScpB-Fn binding interaction requires multiple adjacent Fn molecules there will be minimal binding at the lowest surface concentrations when there are few clusters of Fn molecules with a unimodal distribution corresponding to the low affinity binding site.…”

“…ScpB has been proposed to interact with adsorbed Fn through two binding sites [22]. One binding site has a high affinity of 4 nM and is expected to be biologically significant.…”