Genetic polymorphisms of group B streptococcus scpB alter functional activity of a cell-associated peptidase that inactivates C5a

Abstract

Many group B Streptococcus agalactiae strains and other pathogenic streptococci express a cell-associated peptidase that inactivates C5a (C5a-ase), the major neutrophil chemoattractant produced by activation of the complement cascade. Type III group B streptococci (GBS) can be classified genotypically into three restriction digest pattern types. Functional C5a-ase activity of GBS correlates with this genetic typing; therefore, we sought to identify a genetic basis for this phenomenon. Southern hybridization confirms that all type III GBS contain scpB, the gene encoding GBS C5a-ase. GBS strains with high C5a-ase functional activity and those with no or very low activity both express immunoreactive C5a-ase. The scpB sequence of strain I30, which has high C5a-ase activity, is 98.2% homologous to the previously reported serotype II GBS scpB sequence. The scpB sequences of strains I25 and GW, which have low or no C5a-ase activity, are identical. The predicted I25 and GW C5a-ase proteins share a four-amino-acid deletion affecting the protease histidine active-site consensus motif. Recombinant I30 C5a-ase has good functional activity, whereas recombinant I25 C5a-ase has low activity. These data demonstrate that functional C5a-ase differences between type III GBS strains are attributable to a genetic polymorphism of scpB. The ubiquitous expression of C5a-ase, irrespective of functional activity, suggests that C5a-ase may have a second, as yet unidentified, function.

FIG. 1

Western blot detection of C5a-ase by MAb F1. Shown is a Western blot of 0.1 μg of rC5a-ase (lane 1) and mutanolysin extracts of strains COH-1 (scpB⁺; lane 2), TOH-97 (scpB isogenic mutant of strain COH-1; lane 3), and TOH-85 (isogenic mutant of csp; lane 4). Positions of molecular mass standards are shown at the left in kilodaltons.

FIG. 2

Southern dot blot analysis of scpB. Five micrograms of genomic DNA from each of 62 type III GBS strains was hybridized with a full-length scpB probe. A gene homologous to scpB was present in all strains tested, including RDP type III-3b strains with markedly reduced or absent functional C5a-ase activity (wells C8 to D10).

FIG. 3

Western blot of C5a-ase. Mutanolysin extracts from strain I30 (high functional C5a-ase activity; lane 1) and strains with absent or low C5a-ase activity (I25, GW, C39, 62059, I32, C35, and 830097; lanes 2 to 7, respectively) were subjected to SDS-PAGE on a 4 to 15% gel, electroblotted to nitrocellulose membranes, and detected with anti-C5a-ase MAb F1. A band corresponding to the predicted size of C5a-ase is detected in mutanolysin extracts from each GBS strain. Positions of molecular mass standards are indicated in kilodaltons.

FIG. 4

C5a-ase functional activity of GBS strains I30, I25, and GW. Shown is the percent adherence of PMNs in the presence of C5a pretreated with 5 × 10⁶, 5 × 10⁷, or 5 × 10⁸ whole GBS. Asterisks indicate significant differences (P < 0.01) between the functional C5a-ase activity of strain I30 and that of strains I25 and GW. Also shown is the percent adhesion of untreated PMNs (left) and PMNs treated with 100 μg of rC5a per ml (right).

FIG. 5

C5a-ase functional activity of mutanolysin extracts from GBS strains I30, I25, and GW. Shown is the percent adherence of PMNs exposed to human C5a pretreated with 1, 5, 25, or 100 μl of mutanolysin extract from strain I30, I25, or GW. Asterisks indicate significant differences between the functional C5a-ase activity of strain I30 and that of strains I25 and GW (∗, P = 0.01; ∗∗, P < 0.01). Also shown is the percent adhesion of untreated PMNs (left) and PMNs treated with 100 μg of rC5a per ml (right).

FIG. 6

Functional activity of recombinant I30 and I25 C5a-ase. Shown is the percent adherence of PMNs incubated with human C5a that has been pretreated with 0.01 to 1 μg of I30 or I25 rC5a-ase per ml. Asterisks indicate significant differences (P < 0.01) in functional C5a-ase activity. Also shown is the percent adhesion of untreated PMNs (left) and PMNs treated with 1 μg of rC5a per ml (right).

FIG. 7

Comparison of the predicted amino acid sequences of the strain I30, I25, and GW scpB to the previously reported GBS C5a-ase amino acid sequence (top row) and the translated sequences of group A streptococcal scpA12 and scpA49 (7, 8). The predicted active sites of the enzyme are indicated by asterisks. A slash indicates the 51-bp deletion present in scpB relative to scpA.

FIG. 7

Comparison of the predicted amino acid sequences of the strain I30, I25, and GW scpB to the previously reported GBS C5a-ase amino acid sequence (top row) and the translated sequences of group A streptococcal scpA12 and scpA49 (7, 8). The predicted active sites of the enzyme are indicated by asterisks. A slash indicates the 51-bp deletion present in scpB relative to scpA.

FIG. 8

Nucleic acid sequences of codons 190 to 207 of the scpB genes from RDP type III-3a (I30, 630640, 560177, I05, and I31) and III-3b (I25, GW, I12, I51, I53, I32, C39, C35, I10, I06, and 861503) strains.