Variation in the Number of Tandem Repeats and Profile of Surface Protein Genes among Invasive Group B Streptococci Correlates with Patient Age

Abstract

The average number of tandem repeats of the rib gene (which encodes the Rib surface protein) in invasive group B streptococci from 29 neonates was smaller than that from 20 adults (6.8 and 8.6, respectively; P < 0.05), implying a distinct contribution of immunity toward this age-related variation.

Group B streptococcus (GBS; Streptococcus agalactiae) is a major pathogen that causes meningitis in neonates and sepsis in nonpregnant adults (3-7) and comprises serotypes Ia, Ib, and II to VIII, based on the capsular polysaccharides (13). The surface proteins of GBS strains are virulence factors and alternative vaccine candidates (11, 14). The alpha-like protein (Alp) family of surface proteins, including alpha C, Rib, Alp2, Alp3, Alp4, and epsilon/Alp1, exhibit a ladder-like pattern when their DNA is amplified by PCR (17). The alpha C protein is often expressed by non-type III GBS isolates, contributes to resistance to opsonophagocytosis, and elicits protective immunity (15), while the Rib protein is mostly expressed by serotype III and confers protective immunity in animal experiments (15). Both proteins exhibit size variability corresponding to the lengths of the tandem repeats in the encoding genes (12); such size variation may arise through recA-independent slipped-strand mispairing during DNA replication (18).

The number of tandem repeats of alpha C is important in the pathogenesis of diseases caused by GBS isolates (15). Several studies have indicated that internal deletion of the bca gene, which encodes the alpha C protein, has been observed in GBS isolates collected from mice pretreated with antiserum and renders an ability to escape from antibodies elicited to the native protein (9, 10, 16); in other words, fewer tandem repeats can offer GBS immune evasion (9). Nevertheless, clinical isolates often contain multiple tandem repeats, suggesting that GBS isolates with large numbers of repeats have enhanced virulence in humans (15). The aims of the present study were to subtype invasive GBS isolates from the bloodstreams of neonates and adults by analysis of the surface protein genes of the Alp family and capsular polysaccharides and to characterize the tandem repeat numbers within the rib gene.

To investigate the genotypes of invasive GBS isolates in southern Taiwan, a total of 156 strains were isolated from the bloodstreams of 42 neonates (ages, ≤90 days) and 114 nonpregnant adults (ages, ≥16 years) at two medical centers from 1994 to 2004. The serotypes of the capsular polysaccharides were determined by using the Group B Streptococci Typing Antisera (Denka Seiken, Nijgata, Japan). Type V strain 2603 V/R and type III strain NEM 316 were used as reference strains for analysis of the serotypes and the surface protein genes. The serotype distributions were as follows: Ia, 17.9%; Ib, 10.3%; II, 4.5%; III, 37.2%; IV, 0.6%; V, 20.5%; VI, 2.6%; and nontypeable, 6.4%. Genes encoding the Alp family of surface proteins, including bca, alp2/3, alp4, rib, and epsilon/alp1, were amplified by multiplex PCR, as described previously (1), with resulting sizes of 398, 334, 110, 295, and 200 bp, respectively. The distributions of the surface protein genes tested were as follows: rib, 31.4%; alp2/3, 26.3%; bca, 26.3%; epsilon/alp1, 10.3%; and none, 5.8%. Most alp2/3 genes (92.7% [38/41]; P = 0.0008, Fisher's test) and bca genes (85.4% [35/41]; P = 0.042, Fisher's test) were harbored in GBS strains from adult patients, while 59.2% (29/49) of the rib genes were in GBS strains from neonates (P < 10⁻⁹, Fisher's test). Only one of these genes was detected in each strain, and alp4 was not detected in our study. Combined with the profiles of the surface protein genes and the capsular polysaccharides, 25 serovariants were obtained. Of the GBS strains collected from adults, serovariants Ib/bca (85.7%; 12/14), III/rib (60%; 15/25), and V/alp2/3 (70%; 21/30) were the three most common subtypes. Only nine subtypes were identified among strains from neonates, with the dominant serovariant III/rib accounting for 87.9% (29/33) of the subtypes. Analysis of potentially age-related correlations by Fisher's test showed that the associations were statistically significant between III/rib and neonatal early-onset infections (53.8%; P = 0.049) and late-onset infections (75.9%; P < 10⁻⁹) and between GBS infections of adults aged 16 to 50 years and Ib/bca (18.8%; P = 0.04) and V/alp2/3 (28.1%; P = 0.019). These findings suggest that specific serovariants could contribute to the pathogenesis of GBS infections in different age groups.

To characterize the tandem repeat numbers in the rib gene of GBS, the full-length gene was amplified from all of the 49 rib-harboring isolates by PCR with primers 5′-CTGAAGTAATTTCAGGAAGTGC-3′ and 5′-ATCCTCTTTTTTCTTAGAAACAGATAA-3′. The number of tandem repeats was determined by subtracting the size of the nonrepeat region from the size of the main PCR product and dividing by a rib repeat size of 237 bp. Our data showed that the number of tandem repeats ranged from 0 to 14, with an average of 7.6. To our knowledge, this is the first report concerning the distribution of the rib gene in invasive GBS isolates. Comparatively, Gravekamp et al. (9) reported that the repeat numbers of the alpha C protein followed a normal distribution, with a mean of 9 to 10.

Although the repeat numbers of the alpha C protein are not different between invasive and carriage strains (2), variations in repeat number can alter their antigenicities and protective epitopes (8). This study revealed that the average numbers of repeats in rib genes were 6.8 in invasive GBS strains from neonates and 8.6 in those from adults (P = 0.045, one-tailed Student's t test), and their medians were remarkably different, for a range of 0 to 14 (6 and 9, respectively) (Fig. 1). This indicated that the variation of repeat numbers in rib was age related; variants with few repeats were more often collected from neonates, and variants with larger numbers of repeats were more often collected from adults. For the alpha C protein of GBS isolates collected from two human maternal and neonatal pairs, the latter contain fewer repeats than the former and are less susceptible to opsonophagocytic killing in the presence of alpha C protein-specific antiserum (16). Furthermore, immunogenicity and protective efficacy are inversely related to the number of repeats, and deletion of repeats in the alpha C protein can enhance the pathogenicity of GBS isolates in immune mice (9, 10). Therefore, neonatal isolates with fewer repeats of the rib gene might reflect their advantage under the pressure of maternal immunity. On the other hand, clinical GBS isolates with larger numbers of repeats of the Alp proteins can prevent the elimination attempts of the host (15) and possess a selective advantage.

FIG. 1.

Box plot depicting the distribution of tandem repeat numbers in the rib gene in 49 GBS strains collected from the bloodstreams of 29 neonatal and 20 adult patients. A P value of 0.046 was obtained by the one-tailed t test.

In conclusion, certain serovariants of invasive GBS isolates were highly associated with patient age, and this age-related correlation was also found in the number of tandem repeats of the rib gene, with larger numbers of repeats present in adult isolates and fewer repeats present in neonatal isolates. This variation in repeat numbers can be attributed to the complex interaction of selective forces like bacterial virulence and counterforces like host immunity.