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, 74 (1), 40-8

Role of Hyaluronidase in Subcutaneous Spread and Growth of Group A Streptococcus

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Role of Hyaluronidase in Subcutaneous Spread and Growth of Group A Streptococcus

Clarise Rivera Starr et al. Infect Immun.

Abstract

Group A streptococcus (GAS) depends on a hyaluronic acid (HA) capsule to evade phagocytosis and to interact with epithelial cells. Paradoxically, GAS also produces hyaluronidase (Hyl), an enzyme that cleaves HA. A common assumption is that Hyl digests structurally identical HA in human tissue to promote bacterial spread. We inactivated the gene encoding extracellular hyaluronidase, hylA, in a clinical Hyl(+) isolate. Hyl(+) and an isogenic Hyl(-) mutant were injected subcutaneously into mice with or without high-molecular-weight dextran blue. The Hyl(-) strain produced small lesions with dye concentrated in close proximity. The Hyl(+) strain produced identical lesions, but the dye diffused subcutaneously. However, Hyl(+) bacteria were not isolated from unaffected skin stained by dye diffusion. Thus, Hyl digests tissue HA and facilitates spread of large molecules but is not sufficient to cause subcutaneous diffusion of bacteria or to affect lesion size. GAS capsule expression was assayed periodically during broth culture and was reduced in Hyl(+) strains relative to Hyl(-) strains at the onset and the end of active capsule synthesis but not during peak synthesis in mid-exponential phase. Thus, Hyl is not sufficiently active to remove capsule during peak synthesis. To demonstrate a possible nutritional role for Hyl, GAS was shown to grow with N-acetylglucosamine but not d-glucuronic acid (both components of HA) as a sole carbon source. However, only Hyl(+) strains could grow utilizing HA as a sole carbon source, suggesting that Hyl may permit the organism to utilize host HA or its own capsule as an energy source.

Figures

FIG. 1.
FIG. 1.
Inactivation of hylA. (A) hylA and surrounding genes of the GAS chromosome; acoABCL-acetoin dehydrogenase complex, Spy1033 (putative lipoate-protein ligase); and Spy1035 (putative UDP-N-acetylmuramyl tripeptide synthetase). The expanded view of hylA shows the integration of pCRS2 with sites of PCR primer binding. Primers 1 and 2 (5′HylAint and 3′HylAint, respectively) were used to generate the 1.5-kb internal hylA fragment on pCRS2. This sequence in present in all strains and is duplicated when pCRS2 cointegrates. Primers 3 and 4 (5′10403HylA and M13FORUniversal) amplified a 3.0-kp product only when pCRS2 integrated into the chromosome. (B) PCR and 1% agarose gel electrophoretic analysis of DNA from strains 06, 067.1, 067.1rev, 2600, 26007.1, and 26007.1rev. The presence of the 3.0-kb fragment in strains 067.1 and 26007.1 confirms the insertion of pCRS2 into hylA in these strains.
FIG. 2.
FIG. 2.
Cell-associated HA in Hyl+ strain 06 and isogenic Hyl mutant (strain 067.1) during the exponential phase of growth (left panel) and the stationary phase of growth (right panel).
FIG. 3.
FIG. 3.
Appearance of skin after injection of cell-free supernatants combined with dextran blue and injected into the flanks of hairless mice (n = 7). Mice injected with culture supernatants of Hyl+ strain 06 and Hyl+ revertant strain, 0671.rev yielded diffusion of blue dye from site of injection (black circles) compared to Hyl strain. As a positive control for diffusion, bovine testicular hyaluronidase showed diffusion of dye from point of injection. As a negative control, Todd-Hewitt broth alone was injected with dye, which remained localized to site of injection.
FIG. 4.
FIG. 4.
Diffusion of Hyl+ and Hyl streptococci in murine skin. The left panel shows mice injected with 107 bacteria and dextran blue. The blue dye diffused away from the site of injection of a HylA+ strains but not with a HylA mutant. The right panel shows a schematic of punch biopsies taken from mice injected with Hyl+ and Hyl bacteria. At the site of injection, bacteria were found in all mice tested (n = 7). At 10 mm, one mouse infected with Hyl+ strain 06 had bacteria under the skin. At 20 mm, no mice had detectable levels of bacteria under the skin regardless of the bacterial phenotype.
FIG. 5.
FIG. 5.
Growth of strains 06 (Hyl+) and 067.1 (Hyl) in minimal medium with various carbon sources. Both strains were capable of growing in glucose, a combination of the sugars N-acetylglucosamine and d-glucuronic acid and N-acetylglucosamine alone. Only Hyl+ strain was capable of growing in HA alone. Neither strain could grow in d-glucuronic acid alone.

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