Impairment of melibiose utilization in Streptococcus mutans serotype c gtfA mutants.

The Streptococcus mutans serotype c gtfA gene encodes a 55-kilodalton sucrose-hydrolyzing enzyme. Analysis of S. mutans gtfA mutants revealed that the mutant strains were specifically impaired in the ability to use melibiose as a sole carbon source. S. mutans gtfA mutant strains synthesized less alpha-galactosidase activity inducible by raffinose than wild-type strains. Melibiose (an inducer in wild-type strains) failed to induce significant levels of alpha-galactosidase in the mutant strains. We hypothesize that melibiose use by S. mutans requires the interaction of the GtfA enzyme, or another gene product under the control of the gtfA promoter, with other gene product(s) involved in melibiose transport or hydrolysis.     

Expression, purification, and characterization of a novel G protein, SGP, from Streptococcus mutans.

The sgp gene of Streptococcus mutans was recently detected immediately downstream from the dgk gene within the same operon. In this study, the sgp gene was subcloned into the pMAL-c2 vector and SGP (S. mutans G protein) was overexpressed in Escherichia coli as a fusion protein with the maltose-binding protein at a level of 40% of total cellular protein. One-step amylose affinity chromatography purification of this fusion protein yielded a product of approximately 95% purity. SGP was purified from this fusion protein following cleavage with protease factor Xa and DEAE-Sephacel chromatography. In nucleotide binding assays, recombinant SGP showed specific binding for GTP and GDP, but not ATP, CTP, and UTP, and also catalyzed efficient hydrolysis of GTP to GDP. Kinetic studies revealed that the SGP Km value for GTP in this reaction was approximately 5.9 microM. Mg2+ also served as a cofactor of SGP in this reaction. In vivo subcellular localization by immunogold labelling demonstrated that SGP was associated with both membrane and cytoplasmic fractions. SGP not only had structural similarities with other G proteins but also proved to have high-level intrinsic GTPase activity. Therefore, SGP appears to be a new member of the G protein superfamily and may participate in transmembrane signaling in the responses of S. mutans cells to environmental stimuli.     

变异链球菌LuxS基因缺陷株的建立及其耐酸能力的研究

目的 建立LuxS基因缺失的变异链球菌突变菌株,并对突变株的耐酸能力进行研究.方法 以变异链球菌UA159为材料,运用基因重组方法将红霉素抗性基因(Eymr)与LuxS基因上下游区域的2个基因片段按一定顺序重组到质粒载体PUC19的多克隆位点中,获得了具有红霉素抗性的重组质粒,将载体质粒转化到含完整LuxS基因的变异链球菌UA159中,利用红霉素抗性筛选出LuxS基因缺失的突变株.检测变异链球菌LuxS基因突变菌株在不同pH环境下生长情况,并以正常菌株为对照.结果 PCR基因扩增结果显示,突变株LuxS基因已被Eymr基因完全替换,不能再编码合成AI-2(autoinducer 2)信号分子,扩增产物经DNA测序证实筛选得到了LuxS基因缺失的突变株,并且突变株不能诱导V.harveyi BB170的生物发光.与变异链球菌标准菌株相比,LuxS基因突变株的生长情况随着pH的降低受到明显抑制.结论 本研究成功构建LuxS基因缺失的变异链球菌突变株,LuxS感应信号系统参与变异链球菌耐酸能力的调控,LuxS基因缺失菌株耐酸能力降低.     

Construction and Verification of LuxS-negative Mutants of Streptococcus Mutans and the Effect of the Absence of LuxS Gene on the Acid Tolerance

Objective： To knock out the entire Luxs gene of Streptococcus mutans （S mutans） UA159 strain via homologous recombination and construct a Luxs-deleted mutant strain of S. mutans. To study the difference between the acid resistance of S. mutans Ingbritt C international standard strain and the acid resistance of LuxS mutant strain. Methods： Two DNA fragments locating in the upper and downstream of Luxs gene were amplified and a erythromycin resistance gene of PJT10 between them were engineered into PUC19 plasmid for constructing the recombination plasmid pUCluxKO. Electrotransformation of S. mutans cells with pUCluxKO-mutant resulted in isolation of erythromycin resistant S. mutans transformants, which was identified by polymerase chain reaction, V. harveyi BB170 luminescence bioassay and sequencing analysis. Solutions of S. mutans standard strain and LuxS mutant strain with same density were made and cultured at pH 3.5 to 7.0 BHI liquid for the same period. Terminal growth situation was compared. Firstly acidized in pH 5.5 BHI liquid, the two strains were cultured at pH 3.0 BHI liquid. The acid tolerance responses of the two strains were compared. Results ： Restriction endonuclease analyses showed that pUCluxKO-mutant vector had been successfully recombined. The Luxsdeleted status of S. mutans mutants was confirmed by PCR with primers which were specific for the genes of Luxs and Erythromycin resistance. S. mutans mutant can not induce bioluminescence, indiating the mutant had been successfully recombined. After twenty generations of culture, the constructed Chinese S. mutans mutants were confirmed to be stable. Significant difference of aciduricity was observed between S. mutans standard strain and LuxS mutant strain. The acid resistance of standard strain was stronger than that of LuxS mutant strain. The two strains both displayed the capability of acid tolerance responses. Conclusion：The S. mutans gene allelic exchange plasmid is constructed correctively and a Luxs-negative mutants of S. mutans is constructed, which can help to further study the role of Luxs in the pathogenesis of S. mutans. LuxS mutant strain is more sensitive to acid inactivation, but the capability of acid tolerance responses exist still.     

Characteristics and cariogenicity of a fructanase-defective Streptococcus mutants strain.

Polymers of D-fructose produced by a variety of oral bacteria are believed to function as extracellular carbohydrate reserves. Degradation of these polysaccharides in plaque following exhaustion of dietary carbohydrates is thought to contribute to the extent and duration of the acid challenge to the tooth surface and thus to the initiation and progression of dental caries. Streptococcus mutans produces a fructanase, the product of the fruA gene, which is capable of degrading beta(2,6)- and beta(2,1)-linked fructans that are commonly synthesized by dental plaque microorganisms. To evaluate the role of the FruA protein in exopolysaccharide metabolism and to assess the contribution of this enzyme to the pathogenic potential of S. mutans, a fructanase-deficient strain of S. mutans was constructed. Inactivation of a cloned fruA gene was accomplished in Escherichia coli by using a mini-Mu dE transposon, and then an isogenic mutant of S. mutans UA159 was constructed by allelic exchange. Successful inactivation of fruA was confirmed through the use of biochemical assays, Western blotting (immunoblotting) with anti-recombinant FruA antisera, and Southern hybridization. The data indicated that FruA was the only fructan hydrolase produced by S. mutans UA159. Inactivation of fruA had no significant effects on glucosyltransferase or fructosyltransferase activity. In the rat caries model using animals fed a high-sucrose diet and ad libitum, there were no significant differences in the number or severity of smooth surface, sulcal, or root caries elicited by the fruA mutant and the wild-type organism.     

Cariogenicity of a lactate dehydrogenase-deficient mutant of Streptococcus mutans serotype c in gnotobiotic rats.

A lactate dehydrogenase-deficient (Ldh-) mutant of a human isolate of Streptococcus mutans serotype c was tested in a gnotobiotic rat caries model. Compared with the wild-type Ldh-positive (Ldh+) strains, it was significantly (alpha less than or equal to 0.005) less cariogenic in experiments with two different sublines of Sprague-Dawley rats. The Ldh- mutant strain 044 colonized the oral cavity of the test animals to the same extent as its parent strain 041, although its initial implantation was slightly but not significantly (P greater than or equal to 0.2) less. Multiple oral or fecal samples plated on 2,3,5-triphenyltetrazolium indicator medium revealed no evidence of back mutation from Ldh- to Ldh+ in vivo. Both Ldh+ strain 041 and Ldh- strain 044 demonstrated bacteriocinlike activity in vitro against a number of human strains of mutans streptococci representing serotype a (S. cricetus) and serotypes c and e (S. mutans). Serotypes b (S. rattus) and f (S. mutans) and strains of S. mitior, S. sanguis, and S. salivarius were not inhibited. Thus, Ldh mutant strain 044 possesses a number of desirable traits that suggest it should be investigated further as a possible effector strain for replacement therapy of dental caries. These traits include its stability and low cariogenicity in the sensitive gnotobiotic rat caries model, its bacteriocinlike activity against certain other cariogenic S. mutans (but not against more inocuous indigenous oral streptococci), and the fact that it is a member of the most prevalent human serotype of cariogenic streptococci.     

LuxS基因缺失的变形链球菌突变株的构建及鉴定

目的 通过同源重组法构建LuxS基因缺失的变形链球菌(Streptococcus mutans)突变株.方法 运用基因同源重组方法将红霉素抗性基因(Eymr)连接到PCR扩增LuxS基因两端区域产生的2个基因片段之间,并共同插入到pUCl9载体的多克隆位点中,构建出带红霉素抗性标志的缺失突变载体pUCluxKO.将突变载体转化到含完整LuxS基因的突变受体变形链球菌标准株中,红霉素筛选出LuxS基因缺失的变形链球菌突变株,并经PCR、生物荧光检测及DNA测序鉴定.结果 构建的突变载体经限制性内切核酸酶酶切分析显示,产生的条带与设计结果完全一致.PCR方法扩增突变株LuxS和Eymr基因显示,LuxS基因已被完整敲除掉,经生物荧光检测,突变株不能诱导哈氏弧菌(Vibrio harveyi)BBl70的生物发光,说明不能产生信号分子AI-2(autoinducer-2).DNA测序证实筛选得到了LuxS基因缺失的变形链球菌突变株.连续传代培养后证实,变形链球菌LuxS基因突变株具有良好的稳定性.结论 成功构建出LuxS基因缺失的变形链球菌突变株,为研究LuxS基因对变形链球菌致龋毒力的影响奠定了基础.     

无标记的变异链球菌的clpP基因缺陷株的构建

目的 构建无标记的clpP基因缺陷的变异链球菌(简称变链菌)突变株.方法 设计引物PCR扩增大观霉素(Sp)抗性基因,使loxP位点位于Sp抗性基因的两侧,构建出大观霉素抗性基因盒(loxP-Sp-loxP).将clpP基因克隆到pGEM-T-Easy TA载体后,双酶切以去除clpP基因的部分序列,并连入loxP-Sp-loxP,得到clpP基因缺陷的同源重组载体pIB△clpP-Sp.将该载体线性化并电转变链菌标准株,大观霉素筛选得到clpP基因缺陷株.再以温敏质粒pCrePA电转缺陷株,Cre重组酶表达并删除选择标记基因,继而在限制性温度下培养以消除pCrePA,获得无标记的clpP基因缺陷株,并进行PCR及DNA测序鉴定.结果 PCR及DNA测序结果表明clpP基因内部分序列已被删除,且无Sp抗性基因,该部位只留有一个34 bp的loxP位点.结论 在变链菌中成功构建出无标记的clpP基因缺陷株,为进一步研究clpP基因的功能及其在变链菌致龋过程中的作用奠定了基础.     

Identification of virulence determinants for endocarditis in Streptococcus sanguinis by signature-tagged mutagenesis

Streptococcus sanguinis is a gram-positive, facultative anaerobe and a normal inhabitant of the human oral cavity. It is also one of the most common agents of infective endocarditis, a serious endovascular infection. To identify virulence factors for infective endocarditis, signature-tagged mutagenesis (STM) was applied to the SK36 strain of S. sanguinis, whose genome is being sequenced. STM allows the large-scale creation, in vivo screening, and recovery of a series of mutants with altered virulence. Screening of 800 mutants by STM identified 38 putative avirulent and 5 putative hypervirulent mutants. Subsequent molecular analysis of a subset of these mutants identified genes encoding undecaprenol kinase, homoserine kinase, anaerobic ribonucleotide reductase, adenylosuccinate lyase, and a hypothetical protein. Virulence reductions ranging from 2-to 150-fold were confirmed by competitive index assays. One putatively hypervirulent strain with a transposon insertion in an intergenic region was identified, though increased virulence was not confirmed in competitive index assays. All mutants grew comparably to SK36 in aerobic broth culture except for the homoserine kinase mutant. Growth of this mutant was restored by the addition of threonine to the medium. Mutants containing an insertion or in-frame deletion in the anaerobic ribonucleotide reductase gene failed to grow under strictly anaerobic conditions. The results suggest that housekeeping functions such as cell wall synthesis, amino acid and nucleic acid synthesis, and the ability to survive under anaerobic conditions are important virulence factors in S. sanguinis endocarditis.     

Identification and characterization of an autolysin-encoding gene of Streptococcus mutans

We identified a gene (atlA) encoding autolytic activity from Streptococcus mutans Xc. The AtlA protein predicted to be encoded by atlA is composed of 979 amino acids with a molecular weight of 107,279 and has a conserved beta-1,4-N-acetylmuramidase (lysozyme) domain in the C-terminal portion. Sodium dodecyl sulfate extracts of strain Xc showed two major bacteriolytic bands with molecular masses of 107 and 79 kDa, both of which were absent from a mutant with inactivated atlA. Western blot analysis revealed that the 79-kDa band was derived from the 107-kDa peptide by cleavage of its N-terminal portion. The inactivation of atlA resulted in a marked decrease in autolysis and the formation of very long chains of cells compared to the case for the parent strain. Although both the parent and mutant strains formed biofilms in the presence of sucrose, the biofilms formed by the mutant had a sponge-like architecture with large gaps and contained 30% less biomass than those formed by the parent strain. Furthermore, strain Xc formed glucose-dependent, loose biofilms in the absence of sucrose, but the mutant lost this ability. These results suggest that AtlA may play an important role in biofilm formation by S. mutans. The antibody produced against the C-terminal peptide containing the beta-1,4-N-acetylmuramidase domain drastically inhibited the autolytic activity of strain Xc. This inhibition was specific among the oral streptococci to S. mutans. These results indicate that the catalytic domain of AtlA is located at the C terminus, suggesting that further characterization of this domain may provide a means to control cariogenic dental plaque formation.     

Repeated DNA sequence involved in mutations affecting transport of sucrose into Streptococcus mutans V403 via the phosphoenolpyruvate phosphotransferase system.

Mutants of Streptococcus mutans V403 defective in the intracellular sucrose-6-phosphate hydrolase (product of the scrB gene) are sensitive to sucrose because of the intracellular accumulation of the phosphorylated sugar. Using a scrB mutant prepared by allelic exchange, we have isolated and characterized a number of sucrose-resistant revertants. One such mutant was found to lack the ability to transport sucrose into the cell via the phosphoenolpyruvate-dependent sucrose phosphotransferase system (PTS). Genetic analysis of this strain revealed this lesion to be linked to the scrB gene. This was corroborated by the physical demonstration of an insertion mutation very near scrB. Taken together with DNA sequence information (Y. Sato, F. Poy, G. R. Jacobson, and H. K. Kuramitsu, J. Bacteriol. 171:263-271, 1989), our results indicated that all of the mutations characterized were located in the adjoining scrA gene which encodes the membrane-associated, sugar-specific enzyme II (EIIsucrose) component of the sucrose PTS in S. mutans. Biochemically, such a genetic lesion disables the sucrose PTS and prevents sucrose from entering the cell by this system. In this paper, we detail the nature of two independent insertion mutations and conclude them to be the result of duplicative transposition events into the scrA gene. This region of the chromosome was amplified and purified in large quantities by using the polymerase chain reaction. Examination of the amplified DNA revealed that the two independent insertion mutations were composed of sequences that were indistinguishable by size and by restriction site endonuclease maps. Their insertion points in the scrA gene were approximately 200 bp apart. The amplified DNA fragment was also used as a probe to demonstrate the presence of five copies of this element on the S. mutans V403 chromosome. A second strain, S. mutans V310, also was found to carry similarly arranged, multiple copies of this sequence on its chromosome, suggesting a clonal origin of V403 and V310. The small size of this sequence, its presence in multiple copies on the V403 chromosome, and its ability to duplicate itself semiconservatively into remote sites argue compellingly that it is an insertion sequence element. One such insertion mutant, with a defective sucrose PTS, was tested for virulence in rats and was found to cause caries at levels similar to those of the wild-type strain.     

Role of the Streptococcus mutans gtf genes in caries induction in the specific-pathogen-free rat model.

The role of each of the Streptococcus mutans gtf genes coding for glucan synthesis in cariogenesis was evaluated by using strain UA130 in the specific-pathogen-free (SPF) rat model system. Mutants defective in either or both of the genes required for insoluble glucan synthesis, the gtfB and gtfC genes, exhibited markedly reduced levels of smooth-surface carious lesions relative to that of the parental organism. Likewise, the mutant defective in the gtfD gene coding for the glucosyltransferase-S enzyme synthesizing water-soluble glucans also produced significantly fewer smooth-surface lesions than strain UA130. None of these mutations markedly altered the rate of sulcal caries induction relative to that of the parental organism. In addition, a mutant of strain UA130 defective in the gtfA gene was reexamined in the SPF rat model. In contrast to previous results from a gnotobiotic rat system, these mutants also induced significantly fewer smooth-surface carious lesions compared with that by strain UA130. These results suggest that all four genes are important for smooth-surface caries formation. Furthermore, these results are discussed relative to the differences in the diets utilized in the SPF and gnotobiotic rat model systems for assessing the virulence factors of S. mutans.     

Biochemical characterization and evaluation of virulence of a fructosyltransferase-deficient mutant of Streptococcus mutans V403.

The Streptococcus mutans extracellular fructosyltransferase (FTF) enzyme may play a role in the formation of dental caries by synthesizing a fructan polymer that serves as an extracellular storage polysaccharide. We sought to determine if an FTF-deficient strain of S. mutans was less virulent than wild-type cells in a rat animal model system. Cloned ftf gene sequences from S. mutans GS5 were used to generate a defective copy of the ftf gene by inserting into the ftf coding region a DNA fragment which encoded erythromycin resistance. The plasmid which carried the defective ftf construct was introduced into S. mutans V403 by using genetic transformation. This defective construct replaced, by allelic exchange, the wild-type copy of the ftf gene carried on the V403 chromosome. FTF activity assays indicated that the recombinant strain, V1741, was deficient in fructan synthesis. However, extracellular protein preparations from this strain displayed an increased ability to generate glucose polymers (glucans) compared with V403 preparations. Levels of adherence to glass and rat tooth surfaces by strain V1741 were similar to those of the V403 strain. Both strains caused moderate decay on rat tooth surfaces; however, the FTF-deficient strain was less pathogenic compared with the wild-type strain. These results suggest that FTF activity contributes to the pathogenicity of S. mutans V403, possibly by generating extracellular fructans which serve as storage compounds.     

Characterization of the Streptococcus mutans pyruvate formate-lyase (PFL)-activating enzyme gene by complementary reconstitution of the In vitro PFL-reactivating system

The act gene was identified and an act mutant as well as the pfl mutant was constructed in Streptococcus mutans. Pyruvate formate-lyase (PFL) activity was regenerated with the mixture of the respective cell extracts from these mutants by complementary reconstitution of the in vitro reactivating system. The S. mutans act gene encoded the sole enzyme able to activate the PFL protein in this organism.     

Virulence of a spaP mutant of Streptococcus mutans in a gnotobiotic rat model

Streptococcus mutans, the principal etiologic agent of dental caries in humans, possesses a variety of virulence traits that enable it to establish itself in the oral cavity and initiate disease. A 185-kDa cell surface-localized protein known variously as antigen I/II, antigen B, PAc, and P1 has been postulated to be a virulence factor in S. mutans. We showed previously that P1 expression is necessary for in vitro adherence of S. mutans to salivary agglutinin-coated hydroxyapatite as well as for fluid-phase aggregation. Since adherence of the organism is a necessary first step toward colonization of the tooth surface, we sought to determine what effect deletion of the gene for P1, spaP, has on the colonization and subsequent cariogenicity of this organism in vivo. Germ-free Fischer rats fed a diet containing 5% sucrose were infected with either S. mutans NG8 or an NG8-derived spaP mutant strain, PC3370, which had been constructed by allelic exchange mutagenesis. At 1-week intervals for 6 weeks after infection, total organisms recovered from mandibles were enumerated. At week 6, caries lesions also were scored. A significantly lower number of enamel and dentinal carious lesions was observed for the mutant-infected rats, although there was no difference between parent and mutant in the number of organisms recovered from teeth through 6 weeks postinfection. Coinfection of animals with both parent and mutant strains resulted in an increasing predominance of the mutant strain being recovered over time, suggesting that P1 is not a necessary prerequisite for colonization. These data do, however, suggest a role for P1 in the virulence of S. mutans, as reflected by a decrease in the cariogenicity of bacteria lacking this surface protein.     

On the biogenesis of lipid bodies in ancient eukaryotes: synthesis of triacylglycerols by a Toxoplasma DGAT1-related enzyme

In mammalian cells, the main stored neutral lipids are triacylglycerol and cholesteryl esters, which are produced by two related enzymes, acyl-CoA:diacylglycerol acyltransferase (DGAT) and acyl-CoA:cholesterol acyltransferase (ACAT), respectively. Very little is known about the metabolism, intracellular storage and function of neutral lipids in many pathogenic lower eukaryotes. In this paper, we have characterized the activity of an important triacylglycerol synthetic enzyme in the protozoan Toxoplasma gondii. A full-length cDNA and gene encoding a T. gondii DGAT1-related enzyme were identified and designated TgDGAT1. The gene is composed of 15 exons and 14 introns, and encodes a protein with a predicted M(r) 63.5kDa, containing signature motifs characteristic of the DGAT1 family. The native protein migrates at 44kDa under reducing conditions. TgDGAT1 is an integral membrane protein localized to the parasite cortical and perinuclear endoplasmic reticulum, with the C-terminus oriented to the lumen of the organelle. When a Saccharomyces cerevisiae mutant strain lacking neutral lipid production is transformed with TgDGAT1 cDNA, a significant DGAT activity is reconstituted, resulting in triacylglycerol synthesis and biogenesis of cytosolic lipid inclusions, resembling lipid bodies in T. gondii. No production of steryl esters is observed upon TgDGAT1 expression in yeast. In contrast to human DGAT1 lacking fatty acid specificity, TgDGAT1 preferentially incorporates palmitate. Our results indicate that parasitic protozoa are also neutral lipid accumulators and illustrate the first example of the existence of a functional DGAT gene in an ancient eukaryote, demonstrating that diacylglycerol esterification is evolutionarily conserved.     

Effect of lipopolysaccharide and lipid A on mouse liver pyruvate kinase activity.

Several investigators have reported lipid A as the biologically active unit in the lipopolysaccharide (LPS) molecule. To determine if lipid A was responsible for the reported increases in pyruvate kinase, mice were injected with endotoxin from Salmonella typhimurium SR-11, the Re mutant of Salmonella minnesota R 595, and lipid A-bovine serum albumin conjugate. The livers were homogenized and the activity of pyruvate kinase was measured. Similar increases in enzyme were obtained with all three preparations. These data imply that the lipid portion of the LPS molecule was responsible for alterations in host enzyme activity. To further determine if the lipid portion was the active unit, a lipid-degraded endotoxin (endotoxoid) prepared by potassium methylate treatment was inoculated into mice. An initial increase in liver pyruvate kinase activity was observed with all preparations. The marked increase observed at 16 h with the native product and lipid A conjugate was not obtained with the endotoxoid. These experiments extend and confirm previous observations that lipid A is responsible for the effects associated with LPS. Animals tolerant to endotoxin from S. typhimurium SR-11 were challenged with endotoxin from the Re mutant. A significant increase in pyruvate kinase activity was not obtained, suggesting that anti-O antibodies are not important in the development of tolerance.     

Roles of sortase in surface expression of the major protein adhesin P1, saliva-induced aggregation and adherence,and cariogenicity of Streptococcus mutans

Sortase is a newly discovered transpeptidase that covalently links LPXTGX-containing surface proteins to the gram-positive bacterial cell wall. In this study, the sortase gene (srtA) was isolated from Streptococcus mutans NG8 by PCR. The gene encoded a 246-amino-acid protein, including a 40-amino-acid signal peptide. The srtA gene was insertionally inactivated by a tetracycline resistance cassette. P1, a major surface protein adhesin previously shown to anchor to the peptidoglycan by the LPXTGX motif, was secreted into the culture medium by the srtA mutant. In contrast, the wild-type P1 remained cell wall associated. Complementation of the mutant with srtA restored the P1 surface expression phenotype. P1 produced by the mutant, but not that produced by the wild type and the srtA-complemented mutant, was recognized by an antibody raised against the hydrophobic domain and charged tail C terminal to the LPXTGX motif. These results suggest that the failure to anchor P1 to the cell wall is due to the lack of cleavage of P1 at the LPXTGX motif. The srtA mutant was markedly less hydrophobic than the wild type and the complemented mutant. The srtA mutant failed to aggregate in the presence of saliva or salivary agglutinin and adhered poorly to saliva- or salivary agglutinin-coated hydroxylapatite. In rats, the srtA mutant colonized the teeth poorly when sucrose was absent. When sucrose was present, the srtA mutant colonized the teeth but less effectively and induced significantly less caries (P < 0.05) than the wild-type strain. In conclusion, the sortase enzyme in S. mutans is responsible for anchoring P1 to the cell surface and plays a role in modulating the surface properties and cariogenicity of S. mutans.     

Role of lipid rafts and flagellin in invasion of colonic epithelial cells by Shiga-toxigenic Escherichia coli O113:H21

Shiga-toxigenic Escherichia coli (STEC) O113:H21 strains that lack the locus of enterocyte effacement (LEE) efficiently invade eukaryotic cells in vitro, unlike LEE-positive O157:H7 strains. We used a fliC deletion mutant of the O113:H21 STEC strain 98NK2 (98NK2ΔfliC) to show that invasion of colonic epithelial (HCT-8) cells is heavily dependent on production of flagellin, even though adherence to the cells was actually enhanced in the mutant. Flagellin binds and signals through Toll-like receptor 5 (TLR5), but there was no evidence that either TLR5, the adaptor protein myeloid differentiation primary response gene 88 (MyD88), or the serine kinase interleukin-1 receptor-associated kinase (IRAK) were required for invasion of HCT-8 cells by strain 98NK2, as judged by transfection, RNA knockdown, or inhibitor studies. However, pretreatment of cells with anti-asialo-GM1 significantly decreased 98NK2 invasion (by 40.8%), while neuraminidase treatment (which cleaves terminal sialic acid residues, thus converting GM1 into asialo-GM1) significantly increased invasion (by 70.7%). Pretreatment of HCT-8 cells with either the cholesterol-depleting agent methyl-β-cyclodextrin (MβCD) or the tyrosine kinase inhibitor genistein significantly decreased invasion by 98NK2, indicating a potential role for lipid rafts in the invasion mechanism. Confocal microscopy also showed invading 98NK2 colocalized with lipid raft markers caveolin-1 and GM1. Interestingly, anti-asialo-GM1, neuraminidase, MβCD, and genistein have similar effects on the vestigial level of STEC invasion seen for STEC strain 98NK2ΔfliC, indicating that lipid rafts mediate a common step in flagellin-dependent and flagellin-independent cellular invasion.     

变异链球菌葡聚糖结合蛋白D基因失活菌株的构建和鉴定

目的利用基因打靶技术构建变异链球菌葡聚糖结合蛋白D基因（gbpD）失活株,用于葡聚糖结合蛋白D基因功能的研究.方法体外培养变异链球菌UA159菌株并以其基因组为模板,对gbpD基因内部序列进行PCR扩增,连接自杀载体pVA8912,分别用酶切及PCR鉴定;转化变异链球菌UA159株,用PCR及Western blot鉴定.结果经鉴定PCR产物及插入片段大小与预期值相符,且为所需目的基因片段,成功构建了自杀质粒pVA8912-gbpD;经PCR鉴定及Western blot鉴定,gbpD基因失活株基因组中gbpD基因内部成功插入目的片段,且该菌株不表达GbpD蛋白.结论成功构建了用于变异链球菌gbpD基因打靶的自杀质粒和gbpD基因失活株,为该基因功能的研究奠定了基础.