Up until several decades ago and groundbreaking discoveries in the field of molecular microbiology, bacteria were believed to be independently functioning single-celled organisms, living a self-sufficient lifestyle (1). In the mid-20th century several pioneer studies conducted on marine bacteria showed that bacterial cells actually favor living in close proximity to such an extent that the number of the organisms adhering to a firm surface greatly surpasses the number of the free swimming, planktonic organisms in the surrounding liquid media (2, 3). It was further confirmed and is now a well-known fact that bacteria prefer living in complex surface-associated communities which were later named biofilms (4).
Biofilm can be defined as an aggregation of one or more groups of different microorganisms, embedded in a self-produced matrix and adhering to a firm surface. Biofilms are ubiquitous. Bacteria can form them on the greatest variety of surfaces, living or non-living, in humid natural conditions, on medical equipment and living tissue, but also in the most extreme living conditions. Recent studies have described bacterial biofilms found in the extreme subzero temperatures of the Antarctic seawaters, thermal waters ranging from 35 to 50 degrees Celsius and conditions of extreme acidity, high metal content and lack of nutrients (5-7).
Bacterial colonies make up approximately 15-20% of the biofilm volume while the rest is an EPS matrix in which the colonies are embedded, a mixture of different natural polymers, primarily polysaccharides, but also a variety of proteins, glycoproteins, glycolipids, and also nucleic acids (8). The surrounding matrix has many important roles in the lives of its inhabitants. It represents a sort of a safe haven for the bacterial colonies, acting as a ward against sometimes extreme changes of environmental conditions, making it easier to understand why bacteria prefer life in biofilms than that in the form of planktonic organisms (9, 10).
Biofilms can be formed from a single-species bacterial community or, which is more typical, represent a community derived from several different microbial species living an interdependent lifestyle. A fact worth mentioning is that even in the mono-species biofilms, phenotypic heterogeneity exists as a response of the individual bacterial cells to local microenvironment conditions (11). Dental plaque biofilm and biofilm in periodontal diseases are among the best described multi-species biofilms. What characterizes these biofilms is their formation on non-shedding surfaces (teeth, fixed prosthodontic appliances) which subsequently enables the formation of stable and complex bacterial communities.
Bearing all of the presented information on bacterial biofilm communities in mind, an inevitable question arises; how do these communities coordinate their behavior?
A glimpse in the history of quorum sensing
Quorum sensing as a form of bacterial communication was first described a little over 30 years ago in a bioluminescent marine bacteria, Aliivibrio (Vibrio) fisheri (12). Until then social cooperation was thought to be a distinctive feature of ‘higher’, developed organisms. Symbiotic relationship between Aliivibrio fischeri and Hawaiian bobtail squid, Euprymna scolopes, has been studied thoroughly and it was the key finding for understanding how bacterial communication works (13). Hawaiian bobtail squid is a nocturnal hunter living in, as its name suggests, clear Hawaiian shallow coastal waters. Its light organ houses bioluminescent bacteria, Aliivibrio fischeri, producing the right amount of light during the night, squid's active hours thus camouflaging their host. Namely, the light from the moon and the stars would normally make the silhouette of the squid stand out to predators below. By emitting a glow from its underside, the squid mimics ambient night light and virtually has no silhouette and creates no shadow. In turn, the bacteria in the squid's light organ feed on sugar and amino-acids. Aliivibrio fischeri is a planktonic bacterium and its quantities are virtually undetectable in ocean waters. The squid’s circadial pump pumps in and out the water containing Aliivibrio fischeri. Every morning at dawn, the squid disposes of up to 95% of Aliivibrio fischeri from the light organ. Concentration of the bacterial population in the light organ increases during the day and by nighttime, and when the critical bacterial population is restored, the production of light is enabled (12). The unusual yet rhythmic process of turning the light on and off left researchers asking how bacteria know when it is the right time to start producing light and shutting it off.
Quorum, by its definition, is a minimum number of members of a certain assembly required to be present in order to make a certain decision. Bacterial quorum implies a minimum, critical concentration of bacterial cells in a population which leads to common, coordinated gene expression and coordinated response to changes in their environment. Quorum sensing can thus be defined as a regulation of gene expression in accordance with cell-population density.
Bacteria produce and secrete chemical signaling molecule, autoinducers. Extracellular concentration of these molecules increases as a function of bacterial cell density. When bacteria detect the minimal threshold stimulatory concentration of a certain autoinducer they act accordingly and alter their behavior in response to it. Quorum sensing can be divided into 4 steps: 1) intracellular synthesis of the signal molecules, 2) secretion of the molecules, either actively or passively, 3) detection of the signaling molecule and its binding to an inducer and 4) gene transcription activation (Figure 1) (13-15).
Quorum sensing systems
Quorum sensing in Gram-Negative bacteria
The type of quorum sensing system as described in Aliivibrio fischeri is the most common quorum sensing system and has so far been observed in more than 70 bacterial species. Two proteins, LuxI (autoinducer synthase which produces acyl-homoserine lactone autoinducer) and LuxR (autoinducer receptor, which is the DNA-binding transcriptional activator) control the gene expression in Aliivibrio fischeri (13). This type of system, regulated by LuxI/R homologes described for different species and acyl homoserine lactone (AHL) as an autoinducer molecule, is mainly used for intra-species communication (16).
Quorum sensing in Gram-Positive bacteria
Unlike Gram-Negative bacteria, Gram-Positive bacteria use modified oligopeptides in the role of autoinducers which are detected by two-component membrane bound histidine kinase receptors (14).
Autoinducer-2 (AI-2) quorum sensing
While both Gram-Negative and Gram-Positive bacteria possess a certain quorum sensing system which enables them to communicate within their own species, to live in a complex biofilm, in a heterogenic community and it requires communication and behavior coordination in a common language. Autoinducer-2 represents a universal autoinducer molecule that enables inter-species communication and was first discovered as the autoinducer of the second quorum sensing system in the marine bacteria Vibrio harveyi (13, 14). Gene luxS of Vibrio harveyi was identified as the encoder of the synthase enzyme that produces autoinducer-2 while, later on, the genes exhibiting sequence similarity were also found in other Gram-Negative and Gram-Positive bacteria (17, 18).
Quorum sensing and the periodontal pathogens
Although studies conducted to characterize quorum sensing systems and quorum sensing coordinated gene expression in periodontal pathogens have mostly been limited to Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, several reports also identified luxS homologous genes in other pathogens, Fusobacterium nucleatum, Prevotella intermedia and Eikenella corrodens (19, 20). Periodontal pathogens, as well as some other studied oral streptococci species, possess the AI-2 quorum sensing circuit, which enables them inter-species communication in complex bacterial communities of the oral biofilm (19, 21, 22). Acyl homoserine lactone (AHL)-mediated quorum sensing system, similar to the one of Aliivibrio fischeri, has not been found in periodontal pathogens so far (19).
Although microbial etiology in periodontitis is indisputable, disease onset and progression is now associated with population shift within the microbial biofilm community. Periodontal pathogens are overrepresented in samples taken from diseased places, whereas they represent a significantly small portion of the total species in healthy sites (23, 24). The increased complexity and shifts in the microbial community are the result of species association and intra- and inter-species communication.
The role of quorum sensing in biofilm development
The role of quorum sensing in biofilm formation was first studied on non-oral organisms, single-species Pseudomonas aeruginosa community and also biofilm communities comprised of Pseudomonas aeruginosa and Burkholderia species (25). The communication between these bacteria is mediated through AHL signaling quorum sensing systems allowing them to coordinate virulence expression and biofilm formation (26, 27). The ability of Aggregatibacter actinomycetemcomitans to grow in biofilm is closely linked to AI-2 quorum sensing system. A luxS gene mutant (no AI-2 synthase) can form a mature biofilm, yet the biofilm contains notably less biomass. If exogenous autoinducer-2 or a functional luxS gene carrying plasmid is added in the growth medium, normal growth of the biofilm is restored (28). Two periplasmic receptors for autoinducer-2, LsrB and RbsB, were also identified and it was observed that if one or both receptors are inactivated, biofilm growth was either reduced, or completely disabled (29). The fact that Aggregatibacter actinomycetemcomitans also responds to a broad range of autoinducer-2 concentration, is a subject of scientific discussion and further research since the aforementioned periplasmic receptors show different kinetics of interaction with the autoinducer. It seems that RbsB interacts with the autoinducer-2 at higher affinity than LsrB, suggesting that Aggregatibacter actinomycetemcomitans is able to act upon both lower and higher autoinducer-2 concentrations and thus thrive in biofilms with both low and high cell density (29).
Quorum sensing and bacterial iron-uptake
Iron has an essential role in wide variety of bacterial functions since it influences cell composition, intermediary metabolism, secondary metabolism, enzyme activity and many other functions (30). Autoinducer-2 mediated quorum sensing system is also intimately linked to bacterial iron acquisition which was studied on both Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. The Aggregatibacter actinomycetemcomitans can acquire iron from either iron-scavenging chelators (enterobactin-like siderophore) or host cell sources (transferrin and hemoglobin). Autoinducer-2 mediated quorum sensing system has a role in adapting bacteria's iron uptake, depending on the bacterial cell density in the biofilm and autoinducer-2 concentration. Mutant strains exhibit reduced expression of genes coding the receptors for hem, transferrin and hemoglobin, thus reducing iron-uptake from the host cells. Simultaneously, despite the luxS mutation and consequently no production of synthase enzyme and autoinducer-2 respectively, expression of several other genes encoding siderophore receptors is upregulated, inducing a shift towards iron uptake from chelators. This suggests that in a high density cell population, with higher concentration of autoinducer-2, bacteria will obtain iron from host-cell sources, whereas it will turn to iron-scavenging chelators in low autoinducer-2 conditions (31, 32).
Porphyromonas gingivalis obtains iron from hemin (iron-containing porphyrin) by employing specific outer membrane receptors, lipoproteins and proteases (33). It is also controlled by an autoinducer-2 mediated signaling which involves independent regulation of distinct uptake mechanisms. Autoinducer-2 positively or negatively regulates different genes that encode membrane receptors, depending on the available hemin content. There is also a link between iron uptake and virulence in Porphyromonas gingivalis, iron starvation resulting in reduced virulence (34).
Quorum sensing and other functions
AI-2 quorum sensing regulation of stress-related genes in Porphyromonas gingivalis has also been studied. It was shown that LuxS might be involved in promoting survival of Porphyromonas gingivalis in the host by regulating its response to host-induced stresses such as high temperature exposure, resistance to hydrogen peroxide, and changes in the pH by elevated expression of stress proteins (35).
A comparison of the luxS mutant and wild type strains of Aggregatibacter actinomycetemcomitans showed and confirmed autoinducer-2-dependant regulation of genes encoding proteins of the outer membrane, enzymes, transcriptional regulators and fimbriae components. Expression of bacteria's most powerful virulence factor, leukotoxin, reduces trifold after luxS gene inactivation (31).
Quorum sensing inhibitory drugs
Since modern civilization is on the brink of entering a complete antibiotic resistance era, development of quorum sensing inhibitory drugs is sparking more and more interest. By terminating bacterial communication critical aspects of biofilm formation or expression of virulence factors would be affected (36, 37). A variety of quorum sensing systems and structural diversity of autoinducers must also be taken into account. So far, several naturally occurring quorum sensing inhibitory compounds have been discovered and synthetic derivatives based on the known structure of quorum sensing inducers produced. Components of garlic, vanilla extract, pepper and halogenated furanones produced by the marine alga Delisea pulchra showed quorum sensing inhibitory properties (37-44). Studies have shown that addition of ribose derivatives to culture medium negatively affected the formation of Aggregatibacter actinomycetemcomitans biofilm, as autoinducer-2 receptors exhibit homology to known ribose binding proteins, with the ribose acting as a receptor antagonist on autoinducer-2 receptors (45). As one study showed, brominated furanones also influence negatively the biofilm formation of Porphyromonas gingivalis, without affecting the bacterial growth (46). Affecting the autoinducer-2 quorum sensing circuits of the periodontal pathogens as a therapeutic intervention also represents a challenge as other oral bacteria, including the host's beneficial commensals; use these circuits to establish their communities (47).
Quorum sensing regulated gene expression and behavior coordination of the bacterial community points to the fact that bacteria are not solitary organisms as they were once thought of by traditional microbiology. Intra- and inter-species communication is the key in biofilm formation which represents an ever rising problem in healthcare, being the cause of persistent and chronic infections. Although bacterial language is not yet understood in its entirety, the importance of research in this field has been recognized.