< i and Streptococcus mutans" />mylase-binding protein A (AbpA) of S. gordonii, an early ..." />mylase-binding protein A (AbpA) of S. gordonii, an early ..." />mymylase-binding protein A (AbpA) of S. gordonii, an early ..." />mylase-binmylase-binding protein A (Artcut icon" href="//www.ncbi.nlm.nih.gov/favicon.ico" />rtcut icon" rtcut icon" href="//www.ncbi.nlrtcut icon" href="//www.ncbi.nlrtcut icon" href="//www.ncbi.nlm.nih.gov/favicon.ico" /> rtcut irtcut rtcut rtcut icon" href="//www.ncbirtcut icon" href="//wwwrtcut icon" href="//www.ncbi.nlm.nihrtcut icon" href="//www.ncbi.nlm.nih.gortcut icon" href="//www.ncbi.nlm.nih.gov/fartcut icon" href="//www.ncbi.nlm.nih.gov/favicrtcut icon" href="//www.ncbi.nlm.nih.gov/favicon.icortcut icon" href="//www.ncbi.nlm.nih.gov/favicon.ico" />rtcut icon" href="//www.ncbi.nlm.nirtcut irtcurtcutrtcut icon" href="//www.ncbi.nlm.nih.gov/favicon.ico" />le="Skip to the content" tabindex="0" accessle="le="Sle="Skip to the content" tabindex="0" accesskey="3">Skip tle="Skip to the content" tabindex="0" accele="le="Sle="Skip to the content" tabindex="0" acchelper-reset ui-ncbimenu-item-first ui-helper-reset">helper-rehelphelpehelper-reset ui-ncbimenu-item-first ui-helper-reset">helper-reset ui-ncbimhelphelpehelpehelper-reset ui-ncbihelper-reset ui-ncbimenu-item-first ui-helper-rhelphelper-helper-reset uihelphelper-reset ui-ncbimenu-item-fhelper-rehelper-resehelper-reset ui-ncbimenu-itemhelper-helper-helper-resehelper-reset ui-ncbi
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Binding of rAbpA and amylase with GtfB. For cogure/F4/" targetgure/F4/"gure/F4/" targgure/F4/" target="figure" rid-figpopup="F4" rid-ob="ob-F4">Figure 4Figure 4
7"7">24]. In the present st7">24]. In7">24]. In the present study, S. mutans Gtf-B in solution interacted with either soluble amylase or with rAbpA with resultant increased enzyme activity. We chose to study Gtf-B since 7">24]. I7">24]. In the present study, S. mutans Gtf-B in solution interacted with either soluble amylase or with rAbpA with resultant increased enzyme activity. We chose to study Gtf-B since it is strongly associated with cariogenicity of S. mutans, it was readily available in pure form, and could potentially interact with AbpA within the multispecies dental plaque biofilm environment. Because the presence of both AbpA and amylase did not synergistically sti7"7">24<7">27">24]. In the7">2424]. In the present study, S. mutans Gtf-B in solution interacted with either soluble amylase or with rAbpA with resultant increased enzyme activity. We chose to study Gt7">24]. 7">24]. In the present study, S. mutans Gtf-B in solution interacted with either soluble amylase or 7"7">24]. In the present study, S. mutans Gtf-B in solution interacted with either7">24]. In t7">24]. In the present s7">24]. I7">24]. In the present study, 7"7">24]. In the present study, S. mutans Gtf-B in solution interacted with either soluble amylase or with rAbpA with resultant increased enzyme activity. We chose to study Gtf-B since it is strongly associated with cariogenicity of S. mutans, it was readily available in pure form, and could potentially interact with AbpA wit7">24]. In t7">24]. In the present study, S. mutans Gtf-B in statag_243226249">28]. The differences noted in results tag_243226249tag_243226249">28]. The differenctag_243226249tag_243226249">28]. The differences noted in results from the various tag_tag_243226249">28]. The diffefound that found that Sfound that S. gordonii strains deficient in AbpA colonized rats fed a sucrose-free starch diet better than their wild-types, and that this differential advantage in colonization was enhanced in ratfofound that S. gordonii strains deficient in AbpA colonized rats fed a sucrose-free starch diet better than their wild-types, and that this differential advfofound that S. gordonii strains deficient in AbpA colonized rats fed a sucrose-free starch diet better than their wild-types, and that this difound that S. gordonii strains deficient in AbpA colonized rats fed a sucrose-free starch diet better tfound that found that S. gordonii strains deficient in AbpA colonized rats fed a sucrose-free starch diet better than their wild-types, and that this differential advantage in colonization was enhanced in rats fed a high sucrose diet [18]. We interpreted these results at that time by suggesting that AbpA may interact with Gtf to reduce its enzymatic activity, consistenfofound that S. gordonii strains deficient in AbpA colonized rats fed a sucrose-free starch diet better than their wild-types, and that this differential advantage in colonization was enhanced in rats fed a high sucrose diet [18]. We interpreted these results at that time by suggesting that AbpA may interact with Gtf to reduce its enzymatic activity, consistent with a previous report [S. gordonifound that S. gordonii strains deficient in AbpA colonized rahe he in vihe in vivo situation. It is possible that additional interactihe in vihe in vivo situation. It is possible that additional interactions that occur in vivo he in vivo in vivo situation. It is possible that additional interactions that occur he in vivohe in vivo situation.he in vivo he in vivo situation. It is possible that additional interactions that occur in vivo influenhe in vivohe in vivhe in vivo situation. It is possible that additional interactions that occur in vivo influence colonization. Nevertheless, the present results suggest that a protein secreted by S. gordonii (AbpA), along with a host protein (amylase), may together affect the function of S. mutans

Glucosyltranferase activity assay

Gtf activity was evaluated by both qualitative and quantitative assays. For qualitative assays, activity was determined in polyacrylamide gels as previously described [32]. Briefly, cell free supernatants were run on SDS-PAGE followed by overnight incubation of gel in 3% sucrose. The glucan bands synthesized by the GTF were 0.001250.00125% bromophenol blue, and boiled for 3 min. Subsequently, the precipitate components were resolved on 12% SDS-PAGE, stained with SYPRO RED in 7.5% (v/v) acmounts of free glucose and free fructose in the reaction mixture corresponds to the amount of glucosyl residues transferred to glucan, and thus the transferase activity [momounts of free glucose and free fructose in the reaction mixture corresponmounmountsmounts of free glucose and fremounts of free glucose and free fructose in the reaction mixmountmounts of free glucose and free fructose in the reaction mixture corresponds to the amount of glucosyl residues transferred to glucan, and thus the transferase activity [35]. Means (± S.E.) were used to describe the03bc;g of purified amylase, Gtf-B or rAbpA was added and incubated at room temperature for 2 hr. After washing, the samples were probed with polyclonal anti-Gtf-B (kindly provided by Dr. Ann Vacca-Smith), anti-AbpA (25), or anti-amylase antibodies, as appropriate. The plates were then incubated, washed, and probed with the secondary antibody provided in the ELISA kit. The plates were developed using the kit substrate according to the manufacturer's instructions. Following color development, which was proportional to the amount of target protein interacting with the protein bound to the plate, the OD of the solution was read at 630 nm in a plate reader.03bc03bc;g03bc;g of purified amylase, Gt03bc;g of purifie03bc;03bc;g of purified amylase, Gtf-B or rAbpA was added and incubated at room temperature for 2 hr. After washing, the samples were probed with polyclonal anti-Gtf-B (kindly provided by Dr. Ann Vacca-Smith), anti-AbpA (25), or anti-a0303bc;g of purified amylase, Gtf-B or rAbpA was added and incubated at room temperature for 2 hr. After washing, the samples were probed with pol03bc;g of purifi03bc;g of purified a03bc;g 03bc;g of purified amylase, Gtf-B or rAbpA was added and incubated at room temperature for 2 hr. After washing, the samples were probed with polyclonal anti-Gtf-B (kindly provided by Dr. Ann Vacca-Smith), anti-AbpA (25), or anti-amylase antibodies, as appropriate. The plates were then incubated, washed, and probed with the secondary antibody provided in the ELISA kit. The plates were developed using the kit substrate according to the manufacturer's instructions. Following color development, which was proportional to the amount of target protein interacting with the protein bound to the plate, the OD of the solution was read at 630 nm in a plate reader.

Acknowledgements

We thank Dr. Molakala Reddy and Mr. Paucipacipatecipated in the analysis and interpretation of data, co-dcipated in the analysis and interpretation of data, co-drafted and co-wrote the mancipatcipated in the analysis and interpretation of datacipated in the analysis and interpretation of data, co-drafcipated in tcipated in the analysis and interpretation of data, co-drafted and co-wrote the manuscript. JR purifiecipatecipated in the analysis and interpretation of datacipated in tcipated in the analysiscipated in the acipated icipated in the analysis and intercipatecipatcipatedcipatedcipat

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