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      Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans

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          Abstract

          Background

          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. This study aimed to investigate the effects of these missense mutations of the srtA gene on SrtA enzyme activity in S. mutans.

          Methods

          The 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.

          Results

          SrtA △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 ( K m ). However, the catalytic rate constant ( k cat ) and catalytic efficiency ( k cat /K m ) of SrtA △N40(D56E) were reduced compared with those of SrtA △N40(R157H) and SrtA △N40(UA159), whereas the k cat and k cat /K m values of SrtA △N40(R157H) were slightly lower than those of SrtA △N40(UA159).

          Conclusions

          The 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 material

          The online version of this article (doi:10.1186/s12903-016-0204-1) contains supplementary material, which is available to authorized users.

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          Most cited references27

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          Streptococcus adherence and colonization.

          Streptococci readily colonize mucosal tissues in the nasopharynx; the respiratory, gastrointestinal, and genitourinary tracts; and the skin. Each ecological niche presents a series of challenges to successful colonization with which streptococci have to contend. Some species exist in equilibrium with their host, neither stimulating nor submitting to immune defenses mounted against them. Most are either opportunistic or true pathogens responsible for diseases such as pharyngitis, tooth decay, necrotizing fasciitis, infective endocarditis, and meningitis. Part of the success of streptococci as colonizers is attributable to the spectrum of proteins expressed on their surfaces. Adhesins enable interactions with salivary, serum, and extracellular matrix components; host cells; and other microbes. This is the essential first step to colonization, the development of complex communities, and possible invasion of host tissues. The majority of streptococcal adhesins are anchored to the cell wall via a C-terminal LPxTz motif. Other proteins may be surface anchored through N-terminal lipid modifications, while the mechanism of cell wall associations for others remains unclear. Collectively, these surface-bound proteins provide Streptococcus species with a "coat of many colors," enabling multiple intimate contacts and interplays between the bacterial cell and the host. In vitro and in vivo studies have demonstrated direct roles for many streptococcal adhesins as colonization or virulence factors, making them attractive targets for therapeutic and preventive strategies against streptococcal infections. There is, therefore, much focus on applying increasingly advanced molecular techniques to determine the precise structures and functions of these proteins, and their regulatory pathways, so that more targeted approaches can be developed.
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            Sortases and the art of anchoring proteins to the envelopes of gram-positive bacteria.

            The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis.
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              The classification of amino acid conservation.

              A classification of amino acid type is described which is based on a synthesis of physico-chemical and mutation data. This is organised in the form of a Venn diagram from which sub-sets are derived that include groups of amino acids likely to be conserved for similar structural reasons. These sets are used to describe conservation in aligned sequences by allocating to each position the smallest set that contains all the residue types brought together by alignment. This minimal set assignment provides a simple way of reducing the information contained in a sequence alignment to a form which can be analysed by computer yet remains readable.
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                Author and article information

                Contributors
                pelion@163.com
                Yuu_LX@163.com
                taoye18@aliyun.com
                zhouy10.3@163.com
                zhiqinghui@hotmail.com
                lin_hc@163.net
                Journal
                BMC Oral Health
                BMC Oral Health
                BMC Oral Health
                BioMed Central (London )
                1472-6831
                11 April 2016
                11 April 2016
                2016
                : 16
                : 47
                Affiliations
                [ ]Department of Preventive Dentistry, Guanghua School of Stomatology, Sun Yat-Sen University, 56 Ling Yuan Road West, Guangzhou, China
                [ ]Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-Sen University, Guangzhou, China
                [ ]Department of Stomatology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Road West, Guangzhou, China
                Article
                204
                10.1186/s12903-016-0204-1
                4827206
                27068451
                942674ea-6a06-474b-be10-d0b8208f56e6
                © Zhuang et al. 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 26 August 2015
                : 1 April 2016
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: Grant No. 81271123
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2016

                Dentistry
                caries,missense mutation,srta,streptococcus mutans,enzyme activity
                Dentistry
                caries, missense mutation, srta, streptococcus mutans, enzyme activity

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