Growth characteristics of and virulence factor production by group A Streptococcus during cultivation in human saliva

Abstract

Group A Streptococcus (GAS) commonly infects the human oropharynx, but the initial molecular events governing this process are poorly understood. Saliva is a major component of the innate and acquired immune defense in this anatomic site. Although landmark studies were done more than 60 years ago, investigation of GAS-saliva interaction has not been addressed extensively in recent years. Serotype M1 GAS strain MGAS5005 cultured in human saliva grew to approximately 10(7) CFU/ml and, remarkably, maintained this density for up to 28 days. Strains of several other M-protein serotypes had similar initial growth patterns but did not maintain as high a CFU count during prolonged culture. As revealed by analysis of the growth of isogenic mutant strains, the ability of GAS to maintain high numbers of CFU/ml during the prolonged stationary phase in saliva was dependent on production of streptococcal inhibitor of complement (Sic) and streptococcal pyrogenic exotoxin B (SpeB). During cultivation in human saliva, GAS had growth-phase-dependent production of multiple proven and putative extracellular virulence factors, including Sic, SpeB, streptococcal pyrogenic exotoxin A, Mac protein, and streptococcal phospholipase A(2). Our results clearly show that GAS responds in a complex fashion to growth in human saliva, suggesting that the molecular processes that enhance colonization and survival in the upper respiratory tract of humans are well under way before the organism reaches the epithelial cell surface.

FIG. 1.

Schematic of the experimental strategy used to analyze growth of GAS in human saliva. GAS strains were grown to mid-exponential phase (OD₆₀₀, ∼0.5) in THY broth and diluted 1:50 in saliva medium A. After 4.5 h of growth in saliva medium A, a second 1:50 dilution was performed in saliva medium B. Samples were removed every 1.5 h for 9 h and then every 24 h for OD₆₀₀ readings and CFU analysis.

FIG. 2.

Growth patterns of serotype M1 strains in various media. Growth in saliva was assayed by subculturing exponential-growth-phase organisms from THY 1:50 into saliva (saliva medium A). After 4.5 h in saliva medium A, organisms were subcultured 1:50 into saliva medium B. Aliquots were removed at indicated time points, and numbers of CFU were determined after overnight incubation on BSA at 37°C in 5% CO₂. (A) Growth over 7 days for serotype M1 strain MGAS5005. Symbols: Saliva medium A, ▴, dashed line; saliva medium B, ▪, solid line; THY medium, □, dashed line; CDM, •, solid line; saliva plus glucose, ⧫, dashed line; saliva with pH controlled, X, solid line. (B) Growth over 28 days (d) for five serotype M1 strains in saliva medium B. Symbols: MGAS5005, ▪, solid line; SF370, ▴, dashed line; MGAS5474, ▾, solid line; MGAS9415, ⧫, dashed line; MGAS11705, •, solid line. Bars represent standard deviations.

FIG. 3.

Time-kill analyses of serotype M1 strain MGAS5005 in various media. Bacteria were grown in saliva, THY, or CDM, and concentrations were adjusted to achieve a starting inoculum of ∼10⁷ CFU/ml. Ampicillin was then added at indicated concentrations, and viable organisms were assessed by serial dilutions and plating onto BSA. Symbols: Saliva without ampicillin, ▪, solid line; saliva with 0.016 μg/ml ampicillin, ▾, dashed line; saliva with 0.512 μg/ml ampicillin, ▵, solid line; saliva with 1.024 μg/ml ampicillin, •, dashed line; THY with 0.016 ampicillin, □, solid line; CDM with 0.016 μg/ml ampicillin, ⧫, solid line. Bars represent standard deviations.

FIG. 4.

Growth curves of GAS strains in saliva. Growth was assayed by removing aliquots from saliva medium B at indicated time points and counting CFU after overnight incubation on BSA at 37°C in 5% CO₂. (A) Growth curves for serotype M1 strain MGAS5005 (▪, solid line), serotype M3 strain MGAS315 (⧫, dashed line), serotype M6 strain MGAS9645 (○, solid line); serotype M12 strain MGAS9429 (□, dashed line), serotype M28 strain MGAS6180 (•, dashed line), serotype M33 strain MGAS9530 (▴, dashed line), and serotype M73 strain MGAS12083 (▵, solid line). (B) Growth curves for serotype M1 strain MGAS5005 (▪, solid line), serotype M6 strain JRS4 (⧫, dashed line), serotype M6 strain MGAS9645 (○, solid line), serotype M6 strain MGAS10287 (⋄, solid line), serotype M9 strain MGAS12487 (X, dashed line), serotype M43 strain MGAS9464 (▾, dashed line), and serotype M94 strain MGAS12349 (□, dashed line). Results for serotype M11 strain MGAS12292 and serotype M77 strain MGAS9482 overlapped with those for serotype M6 strains MGAS9645 and MGAS10282 and the serotype M9 strain MGAS12487 and are not graphed. Bars represent standard deviations.

FIG. 5.

Western immunoblots showing production of GAS extracellular proteins during growth in saliva. GAS strains were grown in saliva medium B for 4 h, 8 h, and 16 h (corresponding to early exponential, late exponential, and early stationary phase). Proteins present in the supernatant were precipitated, separated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis, and transferred to nitrocellulose. The membranes were probed with primary rabbit antibody (1:10,000) specific for each protein and secondary antibody (1:2,500) conjugated to horseradish peroxidase. Reactivity was visualized with chemiluminescent reagents. Saliva without GAS added was used as a negative control, and the purified protein of interest served as a positive control. Sic was assayed during growth of serotype M1 strain MGAS5005; SlaA was assayed during growth of serotype M3 strain MGAS315; SpeB was assayed during growth of serotype M1 strain MGAS5005 at 4, 8, and 16 h as well as at 7 days and 28 days. The degradation of Sic observed at 16 h has been reported previously (15). N.D., not done.

FIG. 6.

Growth of GAS sic and speB isogenic mutant strains in saliva. Growth was assayed by removing aliquots from saliva medium B at indicated time points and counting CFU after overnight incubation on BSA at 37°C in 5% CO₂. Recombinant Sic and SpeB were added to culture medium as described in Materials and Methods to assess complementation. Symbols: wild-type serotype M1 strain MGAS5005, ▪, solid line; isogenic sic mutant strains, ▾, dashed line; isogenic sic mutant strain with recombinant Sic, ⧫, solid line; isogenic speB mutant strain, ▴, dashed line; isogenic speB mutant strain with recombinant SpeB, □, solid line. Bars represent standard deviations.