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Mutual exclusivity of hyaluronan and hyaluronidase in invasive group A Streptococcus.

Henningham A, Yamaguchi M, Aziz RK, Kuipers K, Buffalo CZ, Dahesh S, Choudhury B, Van Vleet J, Yamaguchi Y, Seymour LM, Ben Zakour NL, He L, Smith HV, Grimwood K, Beatson SA, Ghosh P, Walker MJ, Nizet V, Cole JN - J. Biol. Chem. (2014)

Bottom Line: However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection.Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition.These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Pediatrics, the School of Chemistry and Molecular Biosciences and Australian Infectious Diseases Research Centre, The University of Queensland, St. Lucia, Queensland 4072, Australia.

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A, capsule expression levels of WT clinical M1 GAS isolate 5448 and isogenic ΔhylA mutant. The M1 ΔhylA mutant was complemented with a plasmid expressing the inactive hyaluronidase (HylA) from M1 GAS (pHylA*), or the active HylA from M4 GAS (pHylA). B, capsule expression of WT clinical M4 GAS isolate 4063-05 and isogenic ΔhylA mutant. M4 WT and ΔhylA were transformed with a plasmid expressing the hasABC capsule synthesis operon (pHasABC) or empty vector (pDCerm). C, whole blood survival of nonencapsulated WT M4, encapsulated M4 (M4 pHasABC), nonencapsulated hylA mutant (M4 ΔhylA), encapsulated hylA mutant (M4 ΔhylA pHasABC), encapsulated M1 GAS, and M1 GAS acapsular control (M1 ΔhasA) following a 2-h incubation in whole human blood ex vivo. D, association of His6-tagged C4BPα1–2 with M4 protein in co-precipitation (pull-down) assays. C4BPα1–2 was mixed with M protein in binding buffer for 30 min at 37 °C. Ni2+-nitrilotriacetic acid-agarose beads were added and incubated for 30 min at 37 °C. The beads were washed with binding buffer to remove unbound protein. Bound protein was eluted by boiling in non-reducing sample buffer. Fractions corresponding to unbound and bound protein were resolved by non-reducing SDS-PAGE and visualized with Coomassie stain. E, C4BP binding of nonencapsulated M4 GAS (WT and ΔhylA), encapsulated M4 GAS (M4 pHasABC and ΔhylA pHasABC), and encapsulated WT M1 GAS. F, fibrinogen binding of nonencapsulated M4 GAS (WT and ΔhylA), encapsulated M4 GAS (M4 pHasABC and M4 ΔhylA pHasABC), encapsulated WT M1 GAS, and nonencapsulated M1 GAS (M1 ΔhasA). All values denote arithmetic mean ± S.E. Data were pooled and normalized from 2 independent experiments, each performed in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significantly different.
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Figure 6: A, capsule expression levels of WT clinical M1 GAS isolate 5448 and isogenic ΔhylA mutant. The M1 ΔhylA mutant was complemented with a plasmid expressing the inactive hyaluronidase (HylA) from M1 GAS (pHylA*), or the active HylA from M4 GAS (pHylA). B, capsule expression of WT clinical M4 GAS isolate 4063-05 and isogenic ΔhylA mutant. M4 WT and ΔhylA were transformed with a plasmid expressing the hasABC capsule synthesis operon (pHasABC) or empty vector (pDCerm). C, whole blood survival of nonencapsulated WT M4, encapsulated M4 (M4 pHasABC), nonencapsulated hylA mutant (M4 ΔhylA), encapsulated hylA mutant (M4 ΔhylA pHasABC), encapsulated M1 GAS, and M1 GAS acapsular control (M1 ΔhasA) following a 2-h incubation in whole human blood ex vivo. D, association of His6-tagged C4BPα1–2 with M4 protein in co-precipitation (pull-down) assays. C4BPα1–2 was mixed with M protein in binding buffer for 30 min at 37 °C. Ni2+-nitrilotriacetic acid-agarose beads were added and incubated for 30 min at 37 °C. The beads were washed with binding buffer to remove unbound protein. Bound protein was eluted by boiling in non-reducing sample buffer. Fractions corresponding to unbound and bound protein were resolved by non-reducing SDS-PAGE and visualized with Coomassie stain. E, C4BP binding of nonencapsulated M4 GAS (WT and ΔhylA), encapsulated M4 GAS (M4 pHasABC and ΔhylA pHasABC), and encapsulated WT M1 GAS. F, fibrinogen binding of nonencapsulated M4 GAS (WT and ΔhylA), encapsulated M4 GAS (M4 pHasABC and M4 ΔhylA pHasABC), encapsulated WT M1 GAS, and nonencapsulated M1 GAS (M1 ΔhasA). All values denote arithmetic mean ± S.E. Data were pooled and normalized from 2 independent experiments, each performed in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significantly different.

Mentions: To assess whether an active HylA would have the capacity to digest the capsule of the bacterium, we used precise allelic exchange mutagenesis to delete the hylA gene in M1 GAS strain 5448 (encoding an inactive HylA). Complementation of M1 ΔhylA with a plasmid expressing active HylA from M4 GAS (pHylA), but not the inactive HylA from M1 GAS (pHylA*), completely abolished capsule expression (Fig. 6A). Conversely, to determine whether M4 GAS is capable of synthesizing capsule in the absence of HylA, we constructed a hylA allelic exchange mutant in M4 GAS strain 4063-05, a human blood isolate. Transformation of M4 ΔhylA with pHasABC, a plasmid expressing the hasABC operon from M1 GAS, resulted in capsule expression (Fig. 6B). However, the amount of capsule detected for WT M4 GAS transformed with pHasABC (M4 pHasABC) was significantly less, compared with M4 ΔhylA pHasABC (Fig. 6B). As a corollary, these findings suggest that HylA inactivation prevents capsule degradation in GAS serotypes containing the hasABC operon.


Mutual exclusivity of hyaluronan and hyaluronidase in invasive group A Streptococcus.

Henningham A, Yamaguchi M, Aziz RK, Kuipers K, Buffalo CZ, Dahesh S, Choudhury B, Van Vleet J, Yamaguchi Y, Seymour LM, Ben Zakour NL, He L, Smith HV, Grimwood K, Beatson SA, Ghosh P, Walker MJ, Nizet V, Cole JN - J. Biol. Chem. (2014)

A, capsule expression levels of WT clinical M1 GAS isolate 5448 and isogenic ΔhylA mutant. The M1 ΔhylA mutant was complemented with a plasmid expressing the inactive hyaluronidase (HylA) from M1 GAS (pHylA*), or the active HylA from M4 GAS (pHylA). B, capsule expression of WT clinical M4 GAS isolate 4063-05 and isogenic ΔhylA mutant. M4 WT and ΔhylA were transformed with a plasmid expressing the hasABC capsule synthesis operon (pHasABC) or empty vector (pDCerm). C, whole blood survival of nonencapsulated WT M4, encapsulated M4 (M4 pHasABC), nonencapsulated hylA mutant (M4 ΔhylA), encapsulated hylA mutant (M4 ΔhylA pHasABC), encapsulated M1 GAS, and M1 GAS acapsular control (M1 ΔhasA) following a 2-h incubation in whole human blood ex vivo. D, association of His6-tagged C4BPα1–2 with M4 protein in co-precipitation (pull-down) assays. C4BPα1–2 was mixed with M protein in binding buffer for 30 min at 37 °C. Ni2+-nitrilotriacetic acid-agarose beads were added and incubated for 30 min at 37 °C. The beads were washed with binding buffer to remove unbound protein. Bound protein was eluted by boiling in non-reducing sample buffer. Fractions corresponding to unbound and bound protein were resolved by non-reducing SDS-PAGE and visualized with Coomassie stain. E, C4BP binding of nonencapsulated M4 GAS (WT and ΔhylA), encapsulated M4 GAS (M4 pHasABC and ΔhylA pHasABC), and encapsulated WT M1 GAS. F, fibrinogen binding of nonencapsulated M4 GAS (WT and ΔhylA), encapsulated M4 GAS (M4 pHasABC and M4 ΔhylA pHasABC), encapsulated WT M1 GAS, and nonencapsulated M1 GAS (M1 ΔhasA). All values denote arithmetic mean ± S.E. Data were pooled and normalized from 2 independent experiments, each performed in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significantly different.
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Figure 6: A, capsule expression levels of WT clinical M1 GAS isolate 5448 and isogenic ΔhylA mutant. The M1 ΔhylA mutant was complemented with a plasmid expressing the inactive hyaluronidase (HylA) from M1 GAS (pHylA*), or the active HylA from M4 GAS (pHylA). B, capsule expression of WT clinical M4 GAS isolate 4063-05 and isogenic ΔhylA mutant. M4 WT and ΔhylA were transformed with a plasmid expressing the hasABC capsule synthesis operon (pHasABC) or empty vector (pDCerm). C, whole blood survival of nonencapsulated WT M4, encapsulated M4 (M4 pHasABC), nonencapsulated hylA mutant (M4 ΔhylA), encapsulated hylA mutant (M4 ΔhylA pHasABC), encapsulated M1 GAS, and M1 GAS acapsular control (M1 ΔhasA) following a 2-h incubation in whole human blood ex vivo. D, association of His6-tagged C4BPα1–2 with M4 protein in co-precipitation (pull-down) assays. C4BPα1–2 was mixed with M protein in binding buffer for 30 min at 37 °C. Ni2+-nitrilotriacetic acid-agarose beads were added and incubated for 30 min at 37 °C. The beads were washed with binding buffer to remove unbound protein. Bound protein was eluted by boiling in non-reducing sample buffer. Fractions corresponding to unbound and bound protein were resolved by non-reducing SDS-PAGE and visualized with Coomassie stain. E, C4BP binding of nonencapsulated M4 GAS (WT and ΔhylA), encapsulated M4 GAS (M4 pHasABC and ΔhylA pHasABC), and encapsulated WT M1 GAS. F, fibrinogen binding of nonencapsulated M4 GAS (WT and ΔhylA), encapsulated M4 GAS (M4 pHasABC and M4 ΔhylA pHasABC), encapsulated WT M1 GAS, and nonencapsulated M1 GAS (M1 ΔhasA). All values denote arithmetic mean ± S.E. Data were pooled and normalized from 2 independent experiments, each performed in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significantly different.
Mentions: To assess whether an active HylA would have the capacity to digest the capsule of the bacterium, we used precise allelic exchange mutagenesis to delete the hylA gene in M1 GAS strain 5448 (encoding an inactive HylA). Complementation of M1 ΔhylA with a plasmid expressing active HylA from M4 GAS (pHylA), but not the inactive HylA from M1 GAS (pHylA*), completely abolished capsule expression (Fig. 6A). Conversely, to determine whether M4 GAS is capable of synthesizing capsule in the absence of HylA, we constructed a hylA allelic exchange mutant in M4 GAS strain 4063-05, a human blood isolate. Transformation of M4 ΔhylA with pHasABC, a plasmid expressing the hasABC operon from M1 GAS, resulted in capsule expression (Fig. 6B). However, the amount of capsule detected for WT M4 GAS transformed with pHasABC (M4 pHasABC) was significantly less, compared with M4 ΔhylA pHasABC (Fig. 6B). As a corollary, these findings suggest that HylA inactivation prevents capsule degradation in GAS serotypes containing the hasABC operon.

Bottom Line: However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection.Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition.These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Pediatrics, the School of Chemistry and Molecular Biosciences and Australian Infectious Diseases Research Centre, The University of Queensland, St. Lucia, Queensland 4072, Australia.

Show MeSH
Related in: MedlinePlus