Abstract
BackgroundStreptococcus mutans (S. mutans) is the major aetiological agent of dental caries, and the transpeptidase Sortase A (SrtA) plays a major role in cariogenicity. The T168G and G470A missense mutations in the srtA gene may be linked to caries susceptibility, as demonstrated in our previous studies. This study aimed to investigate the effects of these missense mutations of the srtA gene on SrtA enzyme activity in S. mutans.MethodsThe point mutated recombinant S.mutans T168G and G470A sortases were expressed in expression plasmid pET32a. S. mutans UA159 sortase coding gene srtA was used as the template for point mutation. Enzymatic activity was assessed by quantifying increases in the fluorescence intensity generated when a substrate Dabcyl-QALPNTGEE-Edans was cleaved by SrtA. The kinetic constants were calculated based on the curve fit for the Michaelis-Menten equation.ResultsSrtA△N40(UA159) and the mutant enzymes, SrtA△N40(D56E) and SrtA△N40(R157H), were expressed and purified. A kinetic analysis showed that the affinity of SrtA△N40(D56E) and SrtA△N40(R157H) remained approximately equal to the affinity of SrtA△N40(UA159), as determined by the Michaelis constant (Km). However, the catalytic rate constant (kcat) and catalytic efficiency (kcat/Km) of SrtA△N40(D56E) were reduced compared with those of SrtA△N40(R157H) and SrtA△N40(UA159), whereas the kcat and kcat/Km values of SrtA△N40(R157H) were slightly lower than those of SrtA△N40(UA159).ConclusionsThe findings of this study indicate that the T168G missense mutation of the srtA gene results in a significant reduction in enzymatic activity compared with S. mutans UA159, suggesting that the T168G missense mutation of the srtA gene may be related to low cariogenicity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12903-016-0204-1) contains supplementary material, which is available to authorized users.
Highlights
Streptococcus mutans (S. mutans) is the major aetiological agent of dental caries, and the transpeptidase Sortase A (SrtA) plays a major role in cariogenicity
Expression and purification of SrtA△N40 (UA159) and mutants The SrtA△N40(UA159) and point mutated sortase SrtA△N40(D56E) and SrtA△N40(R157H) were expressed as the recombinant protein that coded by srtA△N120(UA159), srtA△N120(T168G) and srtA△N120 (G470A), respectively
SrtA△N40(UA159) and SrtA△N40(R157H) catalysed the sorting reaction more efficiently than SrtA△N40(D56E), as Discussion S. mutans is the primary pathogen of dental caries, and because of a variety of different genetic events, S. mutans strains exhibit considerable phenotypic variation and differ in cariogenicity [13]
Summary
Streptococcus mutans (S. mutans) is the major aetiological agent of dental caries, and the transpeptidase Sortase A (SrtA) plays a major role in cariogenicity. The T168G and G470A missense mutations in the srtA gene may be linked to caries susceptibility, as demonstrated in our previous studies. We compared the srtA gene of S. mutans strains isolated from caries-free children and children with high-severity caries. The srtA gene sequences of S. mutans clinical isolates were compared with that of S. mutans UA159 using Variant ReporterTM Software (Applied Biosystems, CA, USA) (accession numbers: KP301259 - KP301500). T168G is a point mutation at the 168th base in the srtA gene; this base was T in S. mutans UA159, while some clinical isolates had a G base substitution at that site. The frequency of mutations at the 168 locus was significantly higher in the caries-free group than in the high-severity caries group. Strains with the locus 470 polymorphism exhibited a significantly higher mutation frequency in the high-severity caries group
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