Fernanda C. Petersen

DNA Binding-Uptake System: a Link between Cell-to-Cell Communication and Biofilm Formation

DNA has recently been described as a major structural component of the extracellular matrix in biofilms. In streptococci, the competence-stimulating peptide (CSP) cell-to-cell signal is involved in competence for genetic transformation, biofilm formation, and autolysis. Among the genes regulated in response to the CSP are those involved in binding and uptake of extracellular DNA. We show in this study that a functional DNA binding-uptake system is involved in biofilm formation. A comGB mutant of Streptococcus mutans deficient in DNA binding and uptake, but unaffected in signaling, showed reduced biofilm formation. During growth in the presence of DNase I, biofilm was reduced in the wild type to levels similar to those found with the comGB mutant, suggesting that DNA plays an important role in the wild-type biofilm formation. We also showed that growth in the presence of synthetic CSP promoted significant release of DNA, with similar levels in the wild type and in the comGB mutant. The importance of the DNA binding-uptake system in biofilm formation points to possible novel targets to fight infections.

Biofilm Mode of Growth of Streptococcus intermedius Favored by a Competence-Stimulating Signaling Peptide

Gram-positive and gram-negative bacteria use quorum sensing to coordinate population behavior. In several streptococci, quorum sensing mediated by competence-stimulating peptides (CSP) is associated with development of competence for transformation. We show here that a synthetic CSP favored the biofilm mode of growth of Streptococcus intermedius without affecting the rate of culture growth.

Furanones, potential agents for preventing Staphylococcus epidermidis biofilm infections?

OBJECTIVES Staphylococcus epidermidis is often associated with biofilm infections related to medical implants. The aim of the present study was to find furanones that decrease biofilm formation without irritative or genotoxic effects, or effects on S. epidermidis growth. METHODS After screening including bioluminescence and biofilm assays, 2 furanones out of 11 were chosen for further studies. MIC values of the two furanones were established to determine whether biofilm inhibition effects were ascribed to inhibition of bacterial growth. To further investigate interference with communication, the effect of the furanones was tested in the presence of the autoinducer-2 precursor (S)-4,5-dihydroxy-2,3-pentanedione. The furanones were tested for possible irritative effects by the Hens egg test chorioallantoic membrane procedure. Finally, potential genotoxic effects in mice were assessed by a membrane array, and effects on global gene expression were investigated by using a microarray representing 30,000 genes of the mouse genome. RESULTS From the bioluminescence assay, 4 furanones out of 11 were chosen for further biofilm analyses. Biofilm formation by S. epidermidis was significantly decreased by the four furanones tested at concentrations not affecting microbial growth. Two furanones were chosen for further studies: one that decreased biofilm statistically more than the others and one containing two bromo substituents. The two furanones were found to be non-irritative and non-genotoxic at the concentrations used. CONCLUSIONS Furanones may inhibit biofilm formation through interference with quorum sensing and thus represent promising agents for protecting surfaces from being colonized by S. epidermidis.

THE BIOFILM CONCEPT: CONSEQUENCES FOR FUTURE PROPHYLAXIS OF ORAL DISEASES?

Biofilm control is fundamental to oral health. Existing oral prophylactic measures, however, are insufficient. The main reason is probably because the micro-organisms involved organize into complex biofilm communities with features that differ from those of planktonic cells. Micro-organisms have traditionally been studied in the planktonic state. Conclusions drawn from many of these studies, therefore, need to be revalidated. Recent global approaches to the study of microbial gene expression and regulation in non-oral micro-organisms have shed light on two-component and quorum-sensing systems for the transduction of stimuli that allow for coordinated gene expression. We suggest interference with two-component and quorum-sensing systems as potential novel strategies for the prevention of oral diseases through control of oral biofilms. Information is still lacking, however, on the genetic regulation of oral biofilm formation. A better understanding of these processes is of considerable importance.

AI-2/LuxS Is Involved in Increased Biofilm Formation by Streptococcus intermedius in the Presence of Antibiotics

ABSTRACT Bacteria utilize quorum-sensing communication to organize their behavior by monitoring the concentration of bacterial signals, referred to as autoinducers (AIs). The widespread detection of AI-2 signals and its enzymatic synthase (LuxS) in bacteria suggests that AI-2 is an inter- and intraspecies communication signal. We have previously shown that antibiotic susceptibility is affected by AI-2 signaling in Streptococcus anginosus. Since chronic infections involve persistent biofilms resilient to antibiotic treatment, we explored the role of AI-2/LuxS in Streptococcus intermedius biofilm formation and cell viability when the organism was exposed to sub-MICs of ampicillin, ciprofloxacin, or tetracycline. The S. intermedius wild type (WT) and its isogenic luxS mutant, strain SI006, were exposed to sub-MICs of ampicillin, ciprofloxacin, or tetracycline. Biofilms were formed on polystyrene discs in microtiter plates. To assess planktonic cell viability, the ATP microbial viability assay was performed and the numbers of CFU were determined. For complementation assays, the AI-2 precursor dihydroxy pentanedione (DPD) was used as a supplement for SI006. Relative luxS expression was quantified by real-time PCR. The sub-MICs of all three antibiotics increased biofilm formation in S. intermedius WT. However, biofilm formation by SI006 was either unaffected or reduced (P ≤ 0.05). Bacterial viability tests of biofilm and planktonic cell cultures indicated that SI006 was more susceptible to antibiotics than the WT. DPD complemented the luxS mutant phenotype. Real-time PCR revealed modest yet significant changes in luxS expression in the presence of antibiotic concentrations that increased biofilm formation. In conclusion, in S. intermedius, AI-2/LuxS was involved in antibiotic susceptibility and increased biofilm formation at sub-MICs of antibiotic.

Purification and functional studies of a potent modified quorum‐sensing peptide and a two‐peptide bacteriocin in Streptococcus mutans

Bacteria use quorum‐sensing signals or autoinducers to communicate. The signals in Gram‐positive bacteria are often peptides activated by proteolytic removal of an N‐terminal leader sequence. While investigating stimulation of antimicrobial peptide production by the Streptococcus mutans synthetic competence stimulating peptide signal (21‐CSP), we found a peptide similar to the 21‐CSP, but lacking the three C‐terminal amino acid residues (18‐CSP). The 18‐CSP was more potent in inducing competence, biofilm formation, and antimicrobial activity than the 21‐CSP. Our results indicate that cleavage of the three C‐terminal residues occurred post export, and was not regulated by the CSP‐signalling system. Deletion of comD encoding the CSP receptor abolished the competence and biofilm responses to the 21‐CSP and the 18‐CSP, suggesting that signal transduction via the ComD receptor is involved in the responses to both CSPs. In S. mutans GS5, beside the 18‐CSP we also purified to homogeneity a two‐peptide bacteriocin which production was stimulated by the 18‐CSP and the 21‐CSP. Partial sequence of the two‐peptide bacteriocin revealed the product of the smbAB genes recently described. We found that the peptide SmbB was slightly different from the deduced sequence, and confirmed the prediction that both peptides constituting SmbAB bacteriocin are post‐translationally modified. SmbAB exhibited antimicrobial activity against 11 species of streptococci, Enterococcus faecalis and Staphylocococcus epidermidis. Taken together, the findings support the involvement of the CSP response in bacteriocin production by streptococci and suggest a novel strategy to potentiate autoinducer activity.

Extracellular Identification of a Processed Type II ComR/ComS Pheromone of Streptococcus mutans

The competence-stimulating peptide (CSP) and the sigX-inducing peptide (XIP) are known to induce Streptococcus mutans competence for genetic transformation. For both pheromones, direct identification of the native peptides has not been accomplished. The fact that extracellular XIP activity was recently observed in a chemically defined medium devoid of peptides, as mentioned in an accompanying paper (K. Desai, L. Mashburn-Warren, M. J. Federle, and D. A. Morrison, J. Bacteriol. 194:3774-3780, 2012), provided ideal conditions for native XIP identification. To search for the XIP identity, culture supernatants were filtered to select for peptides of less than 3 kDa, followed by C(18) extraction. One peptide, not detected in the supernatant of a comS deletion mutant, was identified by tandem mass spectrometry (MS/MS) fragmentation as identical to the ComS C-terminal sequence GLDWWSL. ComS processing did not require Eep, a peptidase involved in processing or import of bacterial small hydrophobic peptides, since eep deletion had no inhibitory effect on XIP production or on synthetic XIP response. We investigated whether extracellular CSP was also produced. A reporter assay for CSP activity detection, as well as MS analysis of supernatants, revealed that CSP was not present at detectable levels. In addition, a mutant with deletion of the CSP-encoding gene comC produced endogenous XIP levels similar to those of a nondeletion mutant. The results indicate that XIP pheromone production is a natural phenomenon that may occur in the absence of natural CSP pheromone activity and that the heptapeptide GLDWWSL is an extracellular processed form of ComS, possibly the active XIP pheromone. This is the first report of direct identification of a ComR/ComS pheromone.

Functional Variation of the Antigen I/II Surface Protein in Streptococcus mutans and Streptococcus intermedius

ABSTRACT Although Streptococcus intermedius and Streptococcus mutans are regarded as members of the commensal microflora of the body, S. intermedius is often associated with deep-seated purulent infections, whereas S. mutans is frequently associated with dental caries. In this study, we investigated the roles of the S. mutans and S. intermedius antigen I/II proteins in adhesion and modulation of cell surface characteristics. By using isogenic mutants, we show that the antigen I/II in S. mutans, but not in S. intermedius, was involved in adhesion to a salivary film under flowing conditions, as well as in binding to rat collagen type I. Binding to human fibronectin was a common function associated with the S. mutans and S. intermedius antigen I/II. Adhesion of S. mutans or S. intermedius to human collagen types I or IV was negligible. Hydrophobicity, as measured by water contact angles, and zeta potentials were unaltered in the S. intermedius mutant. The S. mutans isogenic mutants, on the other hand, exhibited more positive zeta potentials at physiological pH values than did the wild type. The results indicate common and species-specific roles for the antigen I/II in mediating the attachment of S. mutans and S. intermedius to host components and in determining cell surface properties.

Role of hyaluronidase in Streptococcus intermedius biofilm

Streptococcus intermedius is found in biofilms on teeth and as a commensal member of the gastrointestinal and urinary floras, but may also be associated with deep-seated purulent infections and infective endocarditis. S. intermedius produces hyaluronidase, an enzyme that breaks down hyaluronan (HA), a major component of the extracellular matrix of connective tissue. We investigated the involvement of hyaluronidase in S. intermedius biofilm formation and dispersal as well as adhesion to human cells. The hyaluronidase activity and expression of the hyl gene were higher in growth media supplemented with HA. Inactivation of the S. intermedius hyaluronidase resulted in a mutant that formed up to 31 % more biofilm in media supplemented with HA. Hyaluronidase added to the medium caused dispersal of S. intermedius biofilm. Adhesion to epithelial cells was similar in the wild-type and the hyaluronidase mutant. We concluded that hyaluronidase may be important for S. intermedius detachment from biofilms but not for adhesion to epithelial cells. The ability of S. intermedius to detach from the surface and to spread may be crucial in the pathogenicity of this micro-organism.

LuxS-mediated signalling in Streptococcus anginosus and its role in biofilm formation

The autoinducer-2 signal (AI-2) produced by several Gram-positive and Gram-negative bacteria mediates interspecies communication. In this study we were able to identify an orthologue of luxS, required for the synthesis of AI-2 signals, in Streptococcus anginosus. Comparative analyses revealed conserved sequences in the predicted S. anginosus LuxS. Expression of luxS was highest during early exponential growth phase. Compared to other oral streptococci, conditioned media from growth of members of the anginosus group were the most efficient in inducing bioluminescence in Vibrio harveyi, indicative of AI-2 signalling. Disruption of luxS in S. anginosus resulted in a mutant deficient in biofilm formation, whereas no effect on planktonic growth rate was observed under various growth conditions. S. anginosus is part of the human flora found in biofilms of the oral cavity, as well as of the upper respiratory, gastrointestinal and urogenital tracts. Such habitats harbour large varieties of bacterial species, among which cell–cell communication may␣play an important role. S. anginosus has also been associated with purulent infections and cancer in the upper digestive tract. Knowledge about the molecular mechanisms involved in S.␣anginosus communication is important for understanding its commensalism and its pathogenic transition.