Pneumococci Can Become Virulent by Acquiring a New Capsule From Oral Streptococci

Abstract

Pneumococcal conjugate vaccines have been successful, but their use has increased infections by nonvaccine serotypes. Oral streptococci often harbor capsular polysaccharide (PS) synthesis loci (cps). Although this has not been observed in nature, if pneumococcus can replace its cps with oral streptococcal cps, it may increase its serotype repertoire. In the current study, we showed that oral Streptococcus strain SK95 and pneumococcal strain D39 both produce structurally identical capsular PS, and their genetic backgrounds influence the amount of capsule production and shielding from nonspecific killing. SK95 is avirulent in a well-established in vivo mouse model. When acapsular pneumococcus was transformed with SK95 cps, the transformant became virulent and killed all mice. Thus, cps from oral Streptococcus strains can make acapsular pneumococcus virulent, and interspecies cps transfer should be considered a potential mechanism of serotype replacement. Our findings, along with publications from the US Centers for Disease Control and Prevention, highlight potential limitations of the 2013 World Health Organization criterion for studying pneumococcal serotypes carried without isolating bacteria. We show that an oral streptococcal strain, SK95, and a pneumococcal strain, D39, both produce chemically identical capsular PS. We also show that transferring SK95 cps into noncapsulated, avirulent pneumococcus gave it the capacity for virulence in a mouse model.

Keywords: pneumococcus; serotype; vaccine.

Figure 1.

Structure of type 2 capsule polysaccharide (PS). A, Overlay of partial (¹H) Nuclear magnetic resonance (NMR) spectra of capsular PSs from type 2 Streptococcus pneumoniae (D39) (red, top) and Streptococcus oralis (SK95) (blue, bottom) recorded at 50°C. As shown, spectra are identical, but the signals of D39 are broader than those of SK95. Asterisks mark choline signals arising from teichoic acid. To facilitate presentation, the 2 spectral regions are plotted with different vertical scales. B, Structure of capsular PS from S. oralis (SK95) as determined with NMR spectroscopy. C, Amount of capsular PSs from SK95 (blue) and D39 (red) versus elution volumes of an S-500 column. Elution volumes of dextran molecular weight standards are indicated with vertical bars. D, Amount of type 2 capsule (y-axis) bound to the enzyme-linked immunosorbent assay (ELISA) plate at different amounts of PS (x-axis). ELISA wells were coated with antiphosphocholine antibody and captured PSs were determined with a monoclonal antibody (mAb) specific for type 2 PS (Hyp2M2). The mAb supernatant was diluted at 1:320, 1:160, and 1:80, respectively, for D39, SK95, and CWPS1 (teichoic acid) wells. Error bars at the data points indicate standard deviations (error bars for CWPS1 were too small to be seen). Abbreviations: A₄₀₅, absorbance at 405 nm; A₆₃₀, absorbance at 630 nm.

Figure 2.

Type 2 capsule expression in wild type and isogenic strains. A, Flow cytometric analysis of capsule expression. Histograms depict binding of monoclonal antibody (mAb) Hyp2M2 to strains D39 (solid black line) and SK95 (solid gray line). The corresponding dashed lines show control binding (secondary antibody alone). B, Genetic map of the cps region of FG34 (TIGR-JS transformant, with D39 cps) and FG44 (TIGR-JS transformant, with SK95 cps). The 5´ crossover points for FG34 and FG44 are between bases 316624–316729 and 316754–316814 of the TIGR4 genome, respectively (see Supplementary Table 2 for details). Arrows and 4-digit numbers indicate binding positions of the polymerase chain reaction (PCR) primers (Supplementary Table 1); numbers in parentheses, expected sizes of the PCR products; and P and P′, promoter locations. C, Mean fluorescent intensity of type 2 capsule expression for indicated strains. Experiments were performed 6 times, and error bars represent standard errors. *P < .01; †P < .001. D, Sequences of the core promoter regions (black bar) of D39 and SK95 (GenBank accession nos. CP000410.2 [41] and AFUB01000057.1 [13]). Gray- and black-shaded regions indicate core promoter motifs at positions −35 and −10, respectively; ATG, translation start codons of cpsA; and black dots, nucleotide mismatch in the core promoter region between D39 and SK95 cps.

Figure 3.

Nonspecific killing (NSK) and inhibition of phagocytosis. A, NSK of various bacterial strains in the presence of different baby rabbit serum (BRS) concentrations. TIGR-JS and R36A are nonencapsulated control strains. FG34 and FG44 are TIGR-JS transformants with D39 cps and SK95 cps, respectively. SPEC1, TIGR4, and TREP19A are encapsulated pneumococcal isolates [33], and their killing curves lie between FG34 and SPEC6B, and are not shown for the purpose of clarity. B, NSK of bacterial isolates (labeled on x-axis) with HL60 cells treated with OBB alone (white bar), OBB with 10 µmol/L cytochalasin D (Cyto-D) (black bar), or OBB with 1% dimethyl sulfoxide (DMSO) control (gray bar) at 10% BRS. Error bars indicate means of 3 replicates with standard errors. *P < .001.

Figure 4.

In vivo virulence study of D39 and SK95 cps. A, Bacteremia at 6 and 24 hours, and survival of BALB/cJ mice that were intraperitoneally infected with either D39 or SK95 strains (4 mice per group). B, Bacteremia and survival of mice after intraperitoneal infection with acapsular pneumococcus (TIGR-JS, 4 mice/group), pneumococcus strain TIGR4-JS with D39 cps (FG34; 3 mice per group), and TIGR4-JS with SK95 cps (FG44; 3 mice per group). C, Bacteremia and survival of mouse after intraperitoneal infection with TIGR-JS with D39 cps (FG3) or with SK95 cps (FG4). Each group has 5 mice. Infection doses per mice were 10³ colony-forming units (CFUs) of D39, 10⁴ CFUs of SK95, or 10⁵ CFUs of TIGR-JS, FG34, FG44, FG3, and FG4. B, C, For bacteremia, errors bars represent standard errors of the mean, and t test were used. Survival curves were compared using log-rank (Mantel-Cox) tests: P = .045 for D39 versus SK95 (A); P = .01 for both TIGR-JS versus FG34 and TIGR-JS versus FG44 (B); P = .02 for FG34 versus FG44 (C). h, hours.