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Bacteria use quorum sensing (QS) to communicate with each other via secreted small autoinducers produced by individuals. QS allows bacteria to display a unified response that benefits the species during adaptation to environment, colonization, and defense against competitors. In oral streptococci, the CSP-ComDE QS is an inducible DNA damage repair system that is pivotal for bacterial survival. In the oral pathogen Streptococcus mutans, the QS system positively influences the formation of antibiotic persisters, cells that can survive antibiotic attack by entering a non-proliferative state. We recently identified a novel gene, pep299, that is activated in the persister cell fraction induced by QS. In this study, we focused our investigation on the role of pep299, a gene encoding a bacteriocin-like peptide, in the formation of antibiotic persisters. Mutant Δ299, unable to produce Pep299, showed a dramatic reduction in the number of stress-induced persisters. Using a co-culture assay, we showed that cells overproducing pep299 induced the formation of persisters in the mutant, suggesting that Pep299 was actively secreted and detected by neighboring cells. Cells exposed to DNA damage conditions activated the gene expression of pep299. Interestingly, our results suggested that the pep299 gene was also involved in the regulation of a QS-inducible toxin–antitoxin system. Our study suggests that the pep299 gene is at the core of the triggered persistence phenotype in S. mutans, allowing cells to transition into a state of reduced metabolic activity and antibiotic tolerance.

An important factor in the assessment of caries risk is the presence of specific oral microflora, especially Streptococcus mutans. Some S. mutans strains possess proteins capable of binding collagen, such as the Cnm and Cbm proteins. The aim is to determine the presence of S. mutans strains carrying collagen binding proteins in a group of subjects with severe early childhood caries (S-ECC). S. mutans strains isolated from 15 S-ECC children were analyzed for collagen binding domains (cbd) of the cnm (cbd/cnm) and cbm (cbd/cbm) genes and their ability to bind to collagen. S. mutans strains positive for cbd/cnm or cbd/cbm were only found in 3 subjects with the most severe caries profile, with one subject having both cbd/cnm and cbd/cbm, and the other two with one of each. cnm/cbm-positive S. mutans strains bound to collagen substrate more avidly compared with negative S. mutans strains from each of the three groups. Our findings of an association between the presence of the collagen binding domains of the cnm/cbm genes in plaque S. mutans and the most aggressive form of caries profile in children offer a potential strategy to identify an individual’s risk for caries progression. Our study should be replicated in other settings and communities in longitudinal and longer-term studies. Our data offer a potential tool in the caries risk management and assessment in children.

With the increase in life expectancy, reducing the visible signs of skin aging has become a major issue. A reduction in collagen and hyaluronic acid synthesis by fibroblasts is a feature of skin aging. The green seaweed, Ulva intestinalis, is an abundant and rich source of nutrients, especially proteins and peptides. The aim of this study was to assess the potential cosmetic properties of a protein fraction from Ulva intestinalis (PROT-1) containing 51% of proteins and 22% of polysaccharides, and its enzymatic peptide hydrolysates on human dermal fibroblasts. PROT-1 was extracted using a patented acid- and solvent-free process (FR2998894 (B1)). The biochemical characterization and chromatographic analysis showed a main set of proteins (25 kDa). To demonstrate the anti-aging potential of PROT-1, fibroblast proliferation and collagen and hyaluronic acid production were assessed on fibroblast cell lines from donors aged 20 years (CCD-1059Sk) and 46 years (CCD-1090Sk). PROT-1 induced a significant increase in collagen and hyaluronic acid production per cell, and a reduction in cell proliferation without increasing cell mortality. These effects were reversed after protein hydrolysis of PROT-1, showing the central role of proteins in this promising anti-aging property.

Bacteriocins are ribosomally synthesized proteinaceous antibacterial peptides. They selectively interfere with the growth of other bacteria. The production and secretion of bacteriocins confer a distinct ecological advantage to the producer in competing against other bacteria that are present in the same ecological niche. Streptococcus mutans, a significant contributor to the development of dental caries, is one of the most prolific producers of bacteriocins, known as mutacins in S. mutans. In this study, we characterized the locus encoding mutacin B-Ny266, a lantibiotic with a broad spectrum of activity. The chromosomal locus is composed of six predicted operon structures encoding proteins involved in regulation, antimicrobial activity, biosynthesis, modification, transport, and immunity. Mutacin B-Ny266 was purified from semisolid cultures, and two inhibitory peptides, LanA and LanA′, were detected. Both peptides were highly modified. Such modifications include dehydration of serine and threonine and the formation of a C-terminal aminovinyl-cysteine (AviCys) ring. While LanA peptide alone is absolutely required for antimicrobial activity, the presence of LanA′ enhanced the activity of LanA, suggesting that B-Ny266 may function as a two-peptide lantibiotic. The activation of lanAA′ expression is most likely controlled by the conserved two-component system NsrRS, which is activated by LanA peptide but not by LanA′. The chromosomal locus encoding mutacin B-Ny266 was not universally conserved in all sequenced S. mutans genomes. Intriguingly, the genes encoding LanAA′ peptides were restricted to the most invasive serotypes of S. mutans. IMPORTANCE Although dental caries is largely preventable, it remains the most common and costly infectious disease worldwide. Caries is initiated by the presence of dental plaque biofilm that contains Streptococcus mutans, a species extensively characterized by its role in caries development and formation. S. mutans deploys an arsenal of strategies to establish itself within the oral cavity. One of them is the production of bacteriocins that confer a competitive advantage by targeting and killing closely related competitors. In this work, we found that mutacin B-Ny266 is a potent lantibiotic that is effective at killing a wide array of oral streptococci, including nearly all S. mutans strains tested. Lantibiotics produced by oral bacteria could represent a promising strategy to target caries pathogens embedded in dental plaque biofilm.

The oral pathogen Streptococcus mutans communicates using a canonical Gram-positive quorum sensing system, CSP-ComDE. The CSP pheromone already known to be involved in the development of genetic competence positively influences the formation of persisters, dormant variants of regular cells that are highly tolerant to antimicrobial therapy. It is now believed that the persistence phenotype is the end result of a stochastic switch in the expression of toxin-antitoxin (TA) modules. TAs consist of a pair of genes that encode two components, a stable toxin and its cognate labile antitoxin. Transcription analyses revealed that three core genes encoding a putative TA system, called SmuATR, were members of the S. mutans CSP regulon. We hypothesized that S. mutans is using its CSP-ComDE system as a deterministic mechanism for persister formation through the activation of smuATR locus. We showed here that the SmuATR system constitutes a novel tripartite type II TA system in which the smuA and smuT genes encode an antitoxin and a toxin, respectively, while SmuR is a transcriptional repressor involved in the autoregulation of the operon. Ectopic expression of SmuA - SmuT is associated with the CSP-inducible persistence phenotype. In contrast, overexpression of SmuT alone is bactericidal and causes membrane permeabilization. To our knowledge, SmuATR is the first functional chromosomal tripartite TA system shown to be induced by the bacterial quorum sensing system and involved in persister formation.

The aim of this study was to evaluate the potential anticoagulant activity of sulphated polysaccharide-containing extracts of six french edible marine macroalgae. Aqueous extracts of brown (Himanthalia elongata, Laminaria digitata, Ascophyllum nodosum, Fucus vesiculosus), green (Ulva lactuca) and red (Chondrus crispus) macroalgae were prepared and their biochemical properties were determined, including major biomolecules, sulphate and ash contents. The anticoagulant activity of each extract was investigated using different scales from the specific antithrombin-dependent pathway (anti-Xa and anti-IIa) to the intrinsic and/or common (Activated Partial Thromboplastin Time, APTT), extrinsic (Prothrombin Time, PT) or common (Thrombin Time, TT) anticoagulant pathways, and compared with those of commercial anticoagulants, heparin and Lovenox®. Laminaria digitata, Fucus vesiculosus and Chondrus crispus extracts showed a significant APTT anticoagulant capacity, only 5-fold lower than that of Lovenox®, which is a pure low molecular weight heparin used as an anticoagulant agent to prevent pulmonary embolism in patients undergoing surgery.

(1) Background: Brown and red algal sulfated polysaccharides have been widely described as anticoagulant agents. However, data on green algae, especially on the Ulva genus, are limited. This study aimed at isolating ulvan from the green macroalga Ulva rigida using an acid- and solvent-free procedure, and investigating the effect of sulfate content on the anticoagulant activity of this polysaccharide. (2) Methods: The obtained ulvan fraction was chemically sulfated, leading to a doubling of the polysaccharide sulfate content in a second ulvan fraction. The potential anticoagulant activity of both ulvan fractions was then assessed using different assays, targeting the intrinsic and/or common (activated partial thromboplastin time), extrinsic (prothrombin time), and common (thrombin time) pathways, and the specific antithrombin-dependent pathway (anti-Xa and anti-IIa), of the coagulation cascade. Furthermore, their anticoagulant properties were compared to those of commercial anticoagulants: heparin and Lovenox&reg, (3) Results: The anticoagulant activity of the chemically-sulfated ulvan fraction was stronger than that of Lovenox&reg, against both the intrinsic and extrinsic coagulation pathways. (4) Conclusion: The chemically-sulfated ulvan fraction could be a very interesting alternative to heparins, with different targets and a high anticoagulant activity.

Streptococci are among the predominant bacterial species living in the human body. They are normally harmless bacteria, but have the ability to cause diverse infections, ranging from mild (e.g., tooth decay and sore throat) to life-threatening (e.g., endocarditis and meningitis). Streptococci have evolved various means of coping with the deleterious effects of environmental stressors and avoiding the host immune system. Recently, several studies have shown that streptococci colonizing the mouth and upper respiratory tract are able to mount complex stress responses in order to persist and successfully survive competition in their ecological niche. Using a small quorum-sensing peptide pheromone acting as a stress-inducible ‘alarmone‘, oral streptococci synchronize the gene expression of a specific group of cells to coordinate important biological activities.

Streptococcus mutans , a member of the human indigenous oral microbiome, produces a quorum-sensing peptide called the competence-stimulating peptide (CSP) pheromone. We previously demonstrated that S. mutans expresses its CSP pheromone under specific stresses and responds to high levels of CSP by inducing cell death in a fraction of the bacterial population. Streptococci lack the classical SOS response, and the induction of the SigX regulon has been proposed to act as a general stress response in Gram-positive bacteria. We show here that inactivation of SigX abolished the CSP-induced cell death phenotype. Among SigX-regulated genes, SMU.836 (now named lytF Sm ), encoding a conserved streptococcal protein, is a functional peptidoglycan hydrolase involved in CSP-induced cell lysis. We also demonstrated that LytF Sm is most likely a self-acting autolysin, since LytF Sm produced by attacker cells cannot trigger CSP-induced lysis of LytF Sm -deficient target cells present in the same environment. Electron microscopy revealed important morphological changes accompanying autolysis of CSP-induced wild-type cultures that were absent in the LytF Sm -deficient mutant. The LytF Sm promoter was activated in the physiological context of elevated concentrations of the CSP pheromone under stress conditions, such as exposure to heat, hydrogen peroxide, and acid. In a long-term survival assay, the viability of a mutant deficient in LytF Sm autolysin was significantly lower than that observed for the wild-type strain. The results of this study suggest that cell death of S. mutans induced by its quorum-sensing CSP pheromone may represent a kind of altruistic act that provides a way for the species to survive environmental stresses at the expense of some of its cells.

The oral biofilm organism Streptococcus mutans must face numerous environmental stresses to survive in its natural habitat. Under specific stresses, S. mutans expresses the competence-stimulating peptide (CSP) pheromone known to induce autolysis and facilitate the uptake and incorporation of exogenous DNA, a process called DNA transformation. We have previously demonstrated that the CSP-induced CipB bacteriocin (mutacin V) is a major factor involved in both cellular processes. Our objective in this work was to characterize the role of CipB bacteriocin during DNA transformation. Although other bacteriocin mutants were impaired in their ability to acquire DNA under CSP-induced conditions, the Δ cipB mutant was the only mutant showing a sharp decrease in transformation efficiency. The autolysis function of CipB bacteriocin does not participate in the DNA transformation process, as factors released via lysis of a subpopulation of cells did not contribute to the development of genetic competence in the surviving population. Moreover, CipB does not seem to participate in membrane depolarization to assist passage of DNA. Microarray-based expression profiling showed that under CSP-induced conditions, CipB regulated ∼130 genes, among which are the comDE locus and comR and comX genes, encoding critical factors that influence competency development in S. mutans . We also discovered that the CipI protein conferring immunity to CipB-induced autolysis also prevented the transcriptional regulatory activity of CipB. Our data suggest that besides its role in cell lysis, the S. mutans CipB bacteriocin also functions as a peptide regulator for the transcriptional control of the competence regulon.

Biofilms are microbial communities attached to surfaces and encased in an extracellular matrix of microbial origin. They represent the predominant form of microbial life. Biofilms are everywhere and can develop on virtually every natural and man-made surface. Biofilms are also ubiquitous in both normal and pathogenic human processes. Biofilm formation has been demonstrated for numerous pathogens and is clearly one of the main strategies for bacterial survival in a variety of sites within the human body. In almost all instances, the biofilm lifestyle helps bacteria survive and persist within the environment. This review discusses the fundamental biology of microbial biofilm and how biofilms impact the pathogenesis of human infections. The different mechanisms involved in the reduced antimicrobial susceptibility of microorganisms in pathogenic biofilms are discussed in detail in this review. Possible approaches that could be explored in the search for new anti-biofilm strategies to eradicate medically relevant biofilms are also presented.

Summary Horizontal gene transfer through natural DNA transformation is an important evolutionary mechanism among bacteria. Transformation requires that the bacteria are physiologically competent to take and incorporate free DNA directly from the environment. Although natural genetic transformation is a remarkable feature of many naturally competent bacteria, the process is energetically expensive for the cells. Consequently, a tight control of the competence state is necessary. The objective of the present work was to help decipher the molecular mechanisms regulating the escape from the competence state in Streptococcus mutans, the principal etiological agent responsible for tooth decay in humans. Our results showed that the cessation of competence in S. mutans was abrupt, and did not involve the accumulation of a competence inhibitor nor the depletion of a competence activator in the extracellular environment. The competence state was repressed at high cell population density via concomitant repression of sigX gene encoding the master regulator of the competence regulon. Co-culture experiments performed with oral and non-oral bacteria showed that S. mutans assesses its own population density and also the microbial density of its surroundings to regulate its competence escape. Interestingly, neither the intra-species and extra-species quorum-sensing systems nor the other 13 two-component regulatory systems identified in S. mutans were involved in the cell-density-dependent escape of the competence state. Altogether, our results suggest a complex mechanism regulating the competence shut-off involving cell-density-dependent repression of sigX through an as yet undefined system, and possibly SigX protein stability.

Bacteria are considered ‘social’ organisms able to communicate with one another using small hormone-like molecules (pheromones) in a process called quorum-sensing. These signalling molecules increase in concentration as a function of bacterial cell density. For most human pathogens, quorum-sensing is critical for virulence and biofilm formation, and the opportunity to interfere with bacterial quorum-sensing could provide a sophisticated means for manipulating the composition of pathogenic biofilms, and possibly eradicating the infection. Streptococcus mutans is a well-characterized resident of the dental plaque biofilm, and is the major pathogen of dental caries (tooth decay). In S. mutans, its CSP quorum-sensing signalling peptide does not act as a classical quorum-sensing signal by accumulating passively in proportion to cell density. In fact, particular stresses such as those encountered in the oral cavity, induces the production of the CSP pheromone, suggesting that the pheromone most probably functions as a stress-inducible alarmone by triggering the signalling to the bacterial population to initiate an adaptive response that results in different phenotypic outcomes. This mini-review discusses two different CSP-induced phenotypes, bacterial ‘suicide’ and dormancy, and the underlying mechanisms by which S. mutans utilizes the same quorum-sensing signalling peptide to regulate two opposite phenotypes.

International audience; P. fluorescens is responsible for the highest depredation of milk because of its capacity to synthesize extracellular lipase and protease which hydrolyze milk fat and proteins. Several P. fluorescens synthesize an extracellular caseinolytic metalloprotease, called AprX. It is important to rapidly detect the presence of a contamination of raw milk by a strain, especially a P. fluorescens strain, having a high potential of depredation. If standard plate count procedures are often employed, they are time consuming and do not permit to rapidly evaluate the potential of depredation. An alternative method consists to search the aprX gene, but such a method remains of low sensitivity and does not allow evaluating the real potential of depredation of the contaminant. After a milk depredation event, three strains of Pseudomonas spp. (F, 2312 and 2313) have been isolated from a dairy plant. Using molecular and phenotypic approaches, these strains were identified as P. fluorescens strains. Their respective extracellular caseinolytic potential was characterized as well as that of several collection strains of P. fluorescens. It appeared that these strains secreted one protease of about 45 kDa, that their extracellular caseinolytic potential was highly variable for one strain to another and that the one of strain F was the highest. The protease secreted by the strain F was purified and its N-terminal sequence established. It shared 100% identity with the domain 14–34 of extracellular alkaline endoprotease sequences which are called AprX for some of them. Its gene was sequenced as well as that of two collection strains of P. fluorescens having a significant lower extracellular caseinolytic potential. The genomic environment of the aprX gene as well as its expression during the strain growth was investigated. It appears that the difference of extracellular caseinolytic potential which has been observed between the three strains does not mainly result from the AprX sequence/structure but it might rather result from the aprX level of expression.