Quorum sensing LuxS/autoinducer-2 inhibits Enterococcus faecalis biofilm formation ability

Abstract Objective: To investigate the relation between biofilm formation ability and quorum sensing gene LuxS/AI-2. Materials and Methods: Enterococcus faecalis (E. faecalis) standard strain ATCC 29212 was used in the study. Long flanking homology polymerase chain reaction method was used to build the LuxS gene knockout strain. Sequential culture turbidity measurement and CFU counting were used to assess the proliferation ability of E. faecalis after the depletion of LuxS. 96-well plate assay was used to quantify the biofilm formation ability; CLSM was used to observe the attached bacteria areas, while scanning electron microscopy (SEM) was performed to observe biofilm microstructure conditions. Results: LuxS gene knockout strains were successfully constructed and identified. The results showed that proliferation ability of E. faecalis was not affected by the depletion of the luxS gene, and the biofilm formation ability of ΔLuxS 29212 significantly decreased (P<0.05). Conclusions: Collectively, our studies provide the LuxS gene's key role in controlling biofilm formation of E. faecalis, which presented a negative regulation, and furthermore, providing us a possible way to conquer the persistent apical periodontitis.

Effect of luxS overexpression on biofilm formation by Streptococcus mutans / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the effect of quorum sensing luxS gene on biofilm formation through construction of a luxS overexpression strain by Streptococcus mutans (Sm).</p><p><b>METHODS</b>In order to construct pIB-luxS plasmid, the luxS gene fragment amplified by PCR was inserted into the shuttle plasmid pIB169 by corresponding double digests. The pIB-luxS plasmid was linearized electro-transformed into Sm cell and the overexpression strain was selected on chloramphenicol plate and testified by electrophoresis and western blot. The growth rate of both Sm wild type strain and its luxS overexpression strain were observed. Methyl thiazolyl tetrazolium (MTT) assay method was used to compare the biofilm formation quantification by both strains at different time points and containing different sucrose. The structures of the biofilms were observed by using confocal laser scanning microscopy, and biofilm-related gene expressions were investigated by real-time PCR. All experiments were performed in triplicate.</p><p><b>RESULTS</b>The luxS overexpression strain was successfully constructed and confirmed by electrophoresis and Western blotting. The planktonic growth mode of the wild-type and luxS overexpression strain showed no difference, but biofilm formed by Sm overexpression strain was 0.400 ± 0.009 and 0.609 ± 0.041 at 14 and 24 h, higher than the wild type strain biofilm at the same time point (0.352 ± 0.028 and 0.533 ± 0.014, respectively, P < 0.05). After adding 0.125% sucrose, biofilm formed by Sm overexpression strain raised to 1.041 ± 0.038, higher than that by the wild type strain (0.831 ± 0.020, P < 0.05). The biofilm formed by both strains were also increased with the sucrose concentration increase, but there was no difference between them. The overexpression strain aggregated into distinct clusters on structure, genes expression including gtfB, ftf, gbpB, relA, brpA, smu630, comDE, vicR were increased (6.10 ± 0.12, 3.34 ± 0.07, 8.75 ± 0.13, 2.96 ± 0.04, 5.20 ± 0.19, 2.20 ± 0.06, 2.32 ± 0.07 and 10.67 ± 0.57 fold) compared to the wild-type strain (P < 0.05).</p><p><b>CONCLUSIONS</b>Quorum sensing luxS gene can promote the biofilm formation of Sm.</p>

Methyl-metabolism contributes to the LuxS regulation of Streptococcus mutans / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the predominant contribution of methyl-metabolism pathway to the regulation of LuxS of Strecptococcus mutans.</p><p><b>METHODS</b>The differences in biofilm formation and aciduricity of Strecptococcus mutans among the methyl-metabolism-complementation strain (KO-S), the parental wide-type strain (WT) and the luxS null strain (KO) were observed by real-time PCR for monitoring the transcriptional level of genes related to biofilm formation (smu.238, gtfD) and aciduricity (smu.44, smu.46) of the studied strains, methyl thiazolyl tetrazolium (MTT) for quantifying the biofilm of the exhibited strains and confocal laser scanning microscopy for estimating the structure of the biofilm.</p><p><b>RESULTS</b>The transcriptional level of smu.44, smu.46, smu.238, gtfD in WT were 1.289 ± 0.051, 1.694 ± 0.140, 1.565 ± 0.107, 1.667 ± 0.196 respectively; in KO were 1.001 ± 0.045, 1.007 ± 0.151, 1.000 ± 0.021, 1.012 ± 0.196 respectively, downregulated compared with WT (P < 0.05); in KO-S were 4.662 ± 0.091, 5.019 ± 0.258, 3.462±0.029, 3.071 ± 0.136 respectively, upregulated compared both with KO and with WT (P < 0.05). The quantity of biofilms formed by the studied strains were WT (1.592 ± 0.213), KO (0.939 ± 0.029), KO- S (2.177 ± 0.226), KO- P (1.020 ± 0.093), respectively, representing a less quantity by KO and KO-P than WT (P < 0.05) and a more quantity by KO-S than other three stains (P < 0.05). According to the observation of biofilms texture by confocal laser scanning microscopy, the WT biofilm was condensed and even. In contrast, fissures and gaps were found scattered in biofilms of KO, KO-P while lessened in that of KO-S, in which high-density bacterial aggregates were observed. The acid assay indicated a smaller biofilm decrease by WT and KO-S than that by KO and KO- P(P < 0.05).</p><p><b>CONCLUSIONS</b>The methyl- metabolism pathway contributes to LuxS regulation on biofilm formation and auiduricity of Strecptococcus mutans.</p>

Modelación por homología de la proteína Luxs de Porphyromonas gingivalis cepa W83 / Modelling by homology of Luxs protein in Porphyromonas gingivalis strain W83

Antecedentes: En las proteínas no se logra siempre su cristalización, de buen tamaño y de buena calidad para someterla a difracción de rayos X. De tal manera que se abre un campo para el desarrollo de estudios teóricos moleculares y proteínicos, que permiten la representación de las moléculas en tres dimensiones, proporcionando una información espacial para estudiar la interacción entre ligandos y receptores macromoleculares. Materialesy Métodos: Estudio In silico, a partir del análisis de secuencias primarias de seis diferentes proteínas LuxS cristalizadas de diversas bacterias, se seleccionó la proteína 1J6X del Helicobacter pylori, por su similaridad con la secuencia de la proteína LuxS en Porphyromonas gingivalis (P. gingivalis) cepa W83, para producir un modelo por homología de esta proteína, utilizando los programas Sybyl y MOE. Se realizó un acoplamiento con el ligando natural para evaluar la reproducibilidad del modelo en un ambiente biológico. Resultados: Se desarrolló el modelado de la proteína LuxS de P. gingivalis cepa W83, que permite el acercamiento a una estructura que se propone, por la interacción entre la proteína y su ligando natural. El modelo generado con recursos computacionales logró una correcta estructura molecular que aceptó la realización de diversos cálculos. El acoplamiento demostró una cavidad donde se logran diversas posiciones del ligando con buenos resultados. Conclusiones: Se obtuvo un modelo 3D para la proteína LuxS en la P. gingivalis cepa W83 validado por diferentes métodos computacionales con una adecuada reproducibilidad biológica por medio del acoplamiento molecular.

Background: Crystallization is not always achieved for all proteins in a good size and a good quality for X-ray diffraction. So that condition opens a field for the development of theoretical molecular and protein studies allowing the representation of the molecules in 3D, providing spatial information to study the interaction between ligands and macromolecular receptors. Materials and Methods: In silico study from primary sequence analysis of six different proteins LuxS crystallized of several bacteria. 1J6X protein of Helicobacter pylori was selected for its similarity with the LuxS protein sequence in Porphyromonas gingivalis (P. gingivalis) strain W83 to produce a homology model of this protein, using the Sybyl and MOE software. A docking was performed to assess the reproducibility of the model in a biological environment. Results: The LuxS protein modelling of P. gingivalis strain W83 was developed, which allows the approach to a proposed structure for the interaction between the protein and its natural ligand. The model generated with computational resources achieved the correct position and biological behavior by means of developed calculations. The docking showed a cavity in which the ligand adopted several positions with good results. Conclusions: A LuxS protein model was obtained, validated by different methods. This generated a 3D model for LuxS protein in P. gingivalis strain W83 with biological reproducibility by means of molecular docking.

Function of luxS gene in sulfurmetabolism of Streptococcus mutans / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the function of luxS in sulfurmetabolism of Streptococcus mutans (Sm).</p><p><b>METHODS</b>The growth with absorbency (A) of the standards and mutant strains was measured and analyzed in the sulfur-limited defined medium at different periods. The laser scanning confocal microscopy (LSCM) was used to observe and compare the biofilm thickness of the two kinds of strains at different culture conditions.</p><p><b>RESULTS</b>The significant increases in the thickness of mutant strain biofilm and its growth were observed after the addition of cysteine, but did not reach the standards strain levels (P < 0.05). The growth and the biofilm thickness of the mutant strains were (1.301 ± 0.009) and (45.009 ± 0.429) µm. When methionine and S-adenosylhomocysteine of certain concentrations were respectively added, the biofilm thickness and the growth of mutant strain were raised but did not reach the level of the standards strain at 24 h (P < 0.05), but at 48 h they did. When the methionine was added in the mutant strains for 24 h, the biofilm thickness and the growth of mutant strain were (0.448 ± 0.028) and (37.068 ± 2.392) µm, as for the adding of S-adenosylhomocysteine were (0.460 ± 0.005) and (27.343 ± 1.107) µm. When adding the supernatant fluid of standard strains, the biofilm thickness and the growth levels of mutant strain were much higher than those of the standards strain. The biofilm thickness and growth of both kinds of strains decreased after the addition of S-adenosylmethionine.</p><p><b>CONCLUSIONS</b>luxS gene plays not only a role in quorum sensing but also a role in sulfurmetabolism.</p>

Research development of tumor treatment with methionine gamma-lyase / 生物医学工程学杂志

Methionine-dependent increase in tumor cells is a specific metabolic defect. This metabolic defect is also a target for selective treatment of cancer. Studies found that the methionine gamma-lyase (methioninase, L-methionine gamma-lyase) can specificly split the methionine of extracellular and intracellular, so it can strongly inhibit the growth of tumor cells and induce apoptosis of tumor cells. However, no effect on normal cells has been found, so the methionine gamma-lyase may play the anti-tumor role. We also explored in the present study another effect of methionine gamma-lyase as a single agent on DNA methylation levels and DNA synthesis, which may change as a result of deprivation of methionine, and thus may enhance anti-tumor effects. Animal studies and clinical trials showed that a variety of methionine dependent methionine gamma-lyase can eliminate the tumor cells. Therefore, methionine restriction is an effective anticancer strategy. Methionine gamma-lyase has shown great prospects as a new type of gene therapy. This article made a review of it.

Detection and analysis of comE and luxS genes in quorum sensing signal pathway from Streptococcus oralis / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To detect and analyze two important genes, comE and luxS, in quorum sensing signal pathway from Streptococcus oralis (S. oralis).</p><p><b>METHODS</b>The total genomic DNA of S. oralis NH521 (a clinically isolated strain) was firstly extracted. The comE and luxS genes were then amplified by polymerase chain reaction (PCR) and further sequenced. The obtained sequences were compared with related sequences in GenBank.</p><p><b>RESULTS</b>Target bands of both comE and luxS genes were detected by electrophoresis. The obtained gene sequences were similar to the corresponding sequences from another S. oralis strain (luxS, 95.0%; comE, 99.6%); however, comparing to gene sequences of another species Streptococcus mutans, comE was more divergent (12.7%) than luxS gene (74.1%).</p><p><b>CONCLUSION</b>This study successfully amplified and sequenced comE and luxS genes from S. oralis NH521 strain. The luxS gene accumulated more mutations than comE gene did between two S. oralis strains, but comE gene is more divergent than luxS gene between two Streptococcus species.</p>

Detection of luxS gene of quorum sensing signal system in periodontal pathogen / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To detect the presence and distribution of luxS gene in quorum sensing signal system in the periodontal pathogens.</p><p><b>METHODS</b>The total DNA of Porphyromonas gingivalis (Pg), Fusobacterium nucleatum (Fn), Actinobacillus acitinomycetimcomtans (Aa) were extracted. The presence of luxS was detected by polymerase chain reaction (PCR). The products of PCR were detected by electrophoresis, sequenced and identified by a Blast search of the GenBank database.</p><p><b>RESULTS</b>Electrophoresis, sequencing and Blast searching indicated that the PCR products of Pg were highly consistent with the luxS gene in GenBank. The sequencing result of Fn was also identified with the target gene. The PCR product of Aa was the same as reference through electrophoresis.</p><p><b>CONCLUSIONS</b>Pg, Fn, Aa contain luxS gene. Further studies may be required to investigate the functions of luxS in the periodontal pathogens.</p>

Study on the biofilm early formation of Streptococcus mutans luxS gene mutation / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of Streptococcus mutans luxS mutarotation on the early biofilm formation.</p><p><b>METHODS</b>Based on the immobilization of magnetic beads by adherent cells, an assay of biofilm quantitative analysis was developed for the kinetic quantification of biofilm formation in this study. Streptococcus mutans luxS mutant strain was constructed and subject to this biofilm luxS mutant strain were compared.</p><p><b>RESULTS</b>The delta luxS mutant started to form a biofilm from the 6th hour (delta BFI = 2.015), and the delta BFI of luxS mutant increased more quickly than that of the wild type strain, until reaching a complete immobilization of the beads after 10 hours (delta BFI = 7.025). The wild-type strain start to form a biofilm from the 10 th hour (delta BFI = 1.875) and the beads were completely immobilized between 12 and 14 hours.</p><p><b>CONCLUSIONS</b>The luxS mutation can accelerate biofilm on a polystyrene surface during the mid-exponential growth phase. And a luxS-dependent signal may play an important role in the early biofilm formation of Streptococcus mutans.</p>

Influence of luxS of Streptococcus mutans on biofilm of dental plaque / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To construct luxS mutant aften luxS gene of Streptococcus mutans (S. mutans) was knocked out, and examine their ability of biofilm formation.</p><p><b>METHODS</b>A recombinant plasmid containing the flanking fragment of luxS of S. mutans was transformed into S. mutans UA159, and selected by brain heart infusion (BHI) agar medium with kanamicin. The luxS mutant further confirmed via polymerase chain reaction (PCR) and the autoinducer-2 (AI-2) bioluminescence assay of Vibrio harveyi (V. harveyi), and ability of luxS mutant and S. mutans UA159 biofilm formation was examined in different phases, in BHI medium with 1% sucrose and 1% glycose by scanning electron microscopy (SEM).</p><p><b>RESULTS</b>LuxS-deficient S. mutans strains were successfully constructed. Compared with S. mutans UA159, the luxS mutant maintained in BHI medium containing 1% sucrose displayed an apparent defect in biofilm formation, while they showed no significant deviation in BHI medium containing 1% glycose.</p><p><b>CONCLUSION</b>luxS gene in S.mutans can play a role in dental plaque biofilm formation, and the luxS gene is possible to regulate sucrose-dependent biofilm formation.</p>

Detection of quorum-sensing pathway and construction of luxS gene allelic exchange plasmid of Streptococcus mutans / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To detect the AI-2 quorum-sensing pathway and construct the luxS g-ene allelic exchange plasmid of Streptococcus mutans.</p><p><b>METHODS</b>To detect AI-2 pathway in Streptococcus mutans, the Vibrio harveyi BB170 was used as reporter strain. The PCR fragments of the upstream and downstream regions of luxS and the Erythromycin resistance gene were amplified with the primers respectively, and these fragments were ligated into pUC19 vector with double endonuclease reaction sequentially, the ligated DNAs were transformed into Escherichia coli DH5alpha, then the reconstructed plasmids were isolated and identified by restricted endonuclease digestions.</p><p><b>RESULTS</b>Streptococcus mutans Ingbritt C could induce luminescence of BB170, suggesting the presence of AI-2 quorum sensing pathway in Streptococcus mutans, and such stimulatory activity was maximal at the mid-log growth phase. The recombinant plasmid pUCluxKO was digested by PstI-BamHI, and the digest product were 1000 bp and 5000 bp. When the pUCluxKO was digested by BamHI-KpnI, the digest product were 1500 bp and 4500 bp. While it was digested by KpnI-EcoRI, the digest product were 1000 bp and 5000 bp. All PCR product was in a single belt respectively.</p><p><b>CONCLUSIONS</b>The recombinant plasmid was cloned effectively and can be used in the construction of S.mutans luxS mutant.</p>

Construction of Streptococcus mutans luxS gene allelic exchange plasmid / 华西口腔医学杂志

<p><b>OBJECTIVE</b>It is reported that Streptococcus mutans luxS gene may have an important role in the interspecies quorum sensing system. To construct the S. mutans luxS gene knockout mutant, this research aim to construct the luxS gene allelic exchange plasmid.</p><p><b>METHODS</b>The upstream and downstream flank DNA fragments of S. mutans luxS gene (Xup, Xdn)and the E. coli kanamycin resistance gene (Kana) were enriched by pfu DNA polymerase with "nest PCR" methods. These fragments were ligated into pBluescript SK (+) Phagemids vector with double endonuclease reaction sequentially.</p><p><b>RESULTS</b>With endonuclease reaction and DNA sequencing, it was proved that the objective plasmid, Xukd-pbsk, was constructed correctively and the kanamycin resistance gene could be expressed in vitro.</p><p><b>CONCLUSION</b>The S. mutans luxS gene allelic exchange plasmid is constructed correctively in this research and can be used in the future research of S. mutans luxS gene knockout mutant.</p>

Phenotypic analysis of luxS gene deletion mutants and its application in virulence regulation research in group B Streptococcus / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the mechanism of virulence regulation in group B streptococcus (BGS) by studying LuxS-related AI-2 quorum-sensing pathway in GBS.</p><p><b>METHODS</b>luxS gene deletion mutants of GBS (Delta lusX) were characterized by reverse transcription (RT)-PCR, colony immunoblot analysis, growth curve measurement, and cAMP determination. Functional analysis of luxS in the deletion mutants was conducted by bioluminescence assay.</p><p><b>RESULTS</b>Genetic analysis results showed that the luxS deletion in the mutant 515-Delta lusX caused upregulation of scpB gene expression. Phenotypic analysis revealed that, in comparison with the wild strain, 515-Delta lusX mutant grew slowly in DCM media but quickly in THY media. An approximately two-fold decrement in bioluminescence was detected in the luxS deletion mutants as compared with the wild strain.</p><p><b>CONCLUSION</b>This study confirms the importance of LuxS molecule in the AI-2 quorum-sensing pathway in GBS and provides new insights into the virulence regulation mechanism of GBS.</p>