The LiaFSR System Regulates the Cell Envelope Stress Response in Streptococcus mutans

Dental Research Institute, Faculty of Dentistry, University of Toronto, 124 Edward St., Toronto, Ontario M5G1G6, Canada.
Journal of bacteriology (Impact Factor: 2.81). 03/2009; 191(9):2973-84. DOI: 10.1128/JB.01563-08
Source: PubMed


Maintaining cell envelope integrity is critical for bacterial survival, including bacteria living in a complex and dynamic
environment such as the human oral cavity. Streptococcus mutans, a major etiological agent of dental caries, uses two-component signal transduction systems (TCSTSs) to monitor and respond
to various environmental stimuli. Previous studies have shown that the LiaSR TCSTS in S. mutans regulates virulence traits such as acid tolerance and biofilm formation. Although not examined in streptococci, homologs
of LiaSR are widely disseminated in Firmicutes and function as part of the cell envelope stress response network. We describe here liaSR and its upstream liaF gene in the cell envelope stress tolerance of S. mutans strain UA159. Transcriptional analysis established liaSR as part of the pentacistronic liaFSR-ppiB-pnpB operon. A survey of cell envelope antimicrobials revealed that mutants deficient in one or all of the liaFSR genes were susceptible to Lipid II cycle interfering antibiotics and to chemicals that perturbed the cell membrane integrity.
These compounds induced liaR transcription in a concentration-dependent manner. Notably, under bacitracin stress conditions, the LiaFSR signaling system
was shown to induce transcription of several genes involved in membrane protein synthesis, peptidoglycan biosynthesis, envelope
chaperone/proteases, and transcriptional regulators. In the absence of an inducer such as bacitracin, LiaF repressed LiaR-regulated
expression, whereas supplementing cultures with bacitracin resulted in derepression of liaSR. While LiaF appears to be an integral component of the LiaSR signaling cascade, taken collectively, we report a novel role
for LiaFSR in sensing cell envelope stress and preserving envelope integrity in S. mutans.

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Available from: Dilani B Senadheera, Aug 14, 2014
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    • "Cell viability was assessed by counting colony forming units (CFUs). The minimum inhibitory concentration (MIC) test was performed according to the broth microdilution method using BHI broth as previously described 29 . Briefly, ∼105 CFU/ml of bacterial cells were added to a 96-well plate containing BHI medium supplemented with twofold serial dilutions of EGCg or CHX. "
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    ABSTRACT: Objective: To test the inhibitory growth activity of green tea catechin incorporated into dental resins compared to resins containing the broad-spectrum antimicrobial compound chlorhexidine against Streptococcus mutans in vitro. Material and Methods: The minimum inhibitory concentrations (MICs) of epigallocatechin-gallate (EGCg) and chlorhexidine (CHX) were determined according to the microdilution method. Resin discs (5 mm x 3 mm) were prepared from Bis-GMA/TEGDMA (R1) and Bis-GMA/CH3Bis-GMA (R2) comonomers (n=9) containing: a) no drug, b) EGCg, c) CHX. Two concentrations of each drug (0.5x MIC and 1x MIC) were incorporated into the resin discs. Samples were individually immersed in a bacterial culture and incubated for 24 h at 37º C under constant agitation. Cell viability was assessed by counting the number of colonies on replica agar plates. Statistical analysis was performed using one-way ANOVA, Tukey and Student t-tests (α=0.05). Results: Both resins containing EGCg and CHX showed a significant inhibition of bacterial growth at both concentrations tested (p<0.05). A significantly higher inhibition was observed in response to resins containing CHX at 0.5x MIC and 1x MIC, and EGCg at 1x MIC when compared to EGCg at 0.5x MIC. Also, EGCg at 0.5x MIC in R1 had a significantly higher growth inhibition than in R2. Conclusions: Both EGCg and CHX retained their antibacterial activity when incorporated into the resin matrix. EGCg at 1x MIC in R1 and R2 resins significantly reduced S. mutans survival at a level similar to CHX. The data generated from this study will provide advances in the field of bioactive dental materials with the potential of improving the lifespan of resin-based restorations.
    Journal of applied oral science: revista FOB 03/2013; 21(2). DOI:10.1590/1678-7757201302430 · 0.92 Impact Factor
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    • "It has also been shown that L. lactis CesSR modulates resistance of peptidoglycan to hydrolysis via induction of spxB [30]. CesSR is homologous to Bacillus subtilis and Streptococcus mutans LiaRS [31], [32] and Staphylococcus aureus VraSR [33], [34], all of which are also involved in the response to cell envelope stresses. In these, and probably in many pathogenic bacteria, this response is linked to resistance to several antibiotics, such as β-lactams and vancomycin. "
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    ABSTRACT: Membrane proteins comprise an important class of molecules whose study is largely frustrated by several intrinsic constraints, such as their hydrophobicity and added requirements for correct folding. Additionally, the complexity of the cellular mechanisms that are required to insert membrane proteins functionally in the membrane and to monitor their folding state makes it difficult to foresee the yields at which one can obtain them or to predict which would be the optimal production host for a given protein. We describe a rational design approach to improve the lactic acid bacterium Lactococcus lactis as a producer of membrane proteins. Our transcriptome data shows that the two-component system CesSR, which senses cell envelope stresses of different origins, is one of the major players when L. lactis is forced to overproduce the endogenous membrane protein BcaP, a branched-chain amino acid permease. Growth of the BcaP-producing L. lactis strain and its capability to produce membrane proteins are severely hampered when the CesSR system itself or particular members of the CesSR regulon are knocked out, notably the genes ftsH, oxaA2, llmg_2163 and rmaB. Overexpressing cesSR reduced the growth defect, thus directly improving the production yield of BcaP. Applying this rationale to eukaryotic proteins, some of which are notoriously more difficult to produce, such as the medically-important presenilin complex, we were able to significantly diminish the growth defect seen in the wild-type strain and improve the production yield of the presenilin variant PS1Δ9-H6 more than 4-fold. The results shed light into a key, and perhaps central, membrane protein quality control mechanism in L. lactis. Modulating the expression of CesSR benefited the production yields of membrane proteins from different origins. These findings reinforce L. lactis as a legitimate alternative host for the production of membrane proteins.
    PLoS ONE 07/2011; 6(7):e21873. DOI:10.1371/journal.pone.0021873 · 3.23 Impact Factor
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    • "oxacillin) of peptidoglycan synthesis. This is a key difference between the VraSR system of S. aureus and the homologous LiaRS systems of other Gram-positive bacteria such as B. subtilis and S. mutans, which are only activated by lipid-II interacting antibiotics, such as bacitracin, ramoplanin and nisin [15-18]. The increased induction spectrum could account for the larger size of the S. aureus CWSS and its protective role against more different classes of antibiotics. "
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    ABSTRACT: Staphylococcus aureus activates a protective cell wall stress stimulon (CWSS) in response to the inhibition of cell wall synthesis or cell envelope damage caused by several structurally and functionally different antibiotics. CWSS induction is coordinated by the VraSR two-component system, which senses an unknown signal triggered by diverse cell wall active agents. We have constructed a highly sensitive luciferase reporter gene system, using the promoter of sas016 (S. aureus N315), which detects very subtle differences in expression as well as measuring > 4 log-fold changes in CWSS activity, to compare the concentration dependence of CWSS induction kinetics of antibiotics with different cell envelope targets. We compared the effects of subinhibitory up to suprainhibitory concentrations of fosfomycin, D-cycloserine, tunicamycin, bacitracin, flavomycin, vancomycin, teicoplanin, oxacillin, lysostaphin and daptomycin. Induction kinetics were both strongly antibiotic- and concentration-dependent. Most antibiotics triggered an immediate response with induction beginning within 10 min, except for tunicamycin, D-cycloserine and fosfomycin which showed lags of up to one generation before induction commenced. Induction characteristics, such as the rate of CWSS induction once initiated and maximal induction reached, were strongly antibiotic dependent. We observed a clear correlation between the inhibitory effects of specific antibiotic concentrations on growth and corresponding increases in CWSS induction kinetics. Inactivation of VraR increased susceptibility to the antibiotics tested from 2- to 16-fold, with the exceptions of oxacillin and D-cycloserine, where no differences were detected in the methicillin susceptible S. aureus strain background analysed. There was no apparent correlation between the induction capacity of the various antibiotics and the relative importance of the CWSS for the corresponding resistance phenotypes. CWSS induction profiles were unique for each antibiotic. Differences observed in optimal induction conditions for specific antibiotics should be determined and taken into account when designing and interpreting CWSS induction studies.
    BMC Microbiology 01/2011; 11(1):16. DOI:10.1186/1471-2180-11-16 · 2.73 Impact Factor
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