Quorum Sensing in Bacteria and a Glance on Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that primarily infects patients with immune deficiency such as neutropenia. Generally, the pathogenesis of Pseudomonas is complex and multifactorial, since the bacterium is both invasive and toxigenic, can cause a range of infections in humans (Table 1) [1-3]. Pseudomonas clearance is often challenging due to immunodeficiency in patients with infection, multidrug resistancy and sometimes β-lactamase production and the inactivation of third-generation cephalosporin [4,5]. P. aeruginosa produces a variety of cellular structures and products involved in the ability to cause disease through enforcing the adhesion, protecting the phagocytosis, modifying the immune response or destroying the host tissue. Generally, the effective factors involved in the development of infection by this bacterium can be divided into two categories: Extracellular virulence factors and structural virulence factors. Extracellular factors include pigments that are active in Fe absorption, prevent the growth of other bacterial species and degrade factors including protease, hemolysins and toxins (Figure 1) [6,7].


Introduction
Pseudomonas aeruginosa is an opportunistic pathogen that primarily infects patients with immune deficiency such as neutropenia. Generally, the pathogenesis of Pseudomonas is complex and multifactorial, since the bacterium is both invasive and toxigenic, can cause a range of infections in humans (Table 1) [1][2][3]. Pseudomonas clearance is often challenging due to immunodeficiency in patients with infection, multidrug resistancy and sometimes β-lactamase production and the inactivation of third-generation cephalosporin [4,5]. P. aeruginosa produces a variety of cellular structures and products involved in the ability to cause disease through enforcing the adhesion, protecting the phagocytosis, modifying the immune response or destroying the host tissue. Generally, the effective factors involved in the development of infection by this bacterium can be divided into two categories: Extracellular virulence factors and structural virulence factors. Extracellular factors include pigments that are active in Fe absorption, prevent the growth of other bacterial species and degrade factors including protease, hemolysins and toxins ( Figure 1) [6,7].
Major structural factors in this bacterium are pili, flagellum and LPS. In addition to these factors, P. aeruginosa has also other capabilities that enhance bacteria pathogenesis, including a broadspectrum Beta-Lactamase (ESBLs) of class A-B and D that is one of by mucoid strains of P. aeruginosa into its surroundings which helps to the attachment and aggregation of bacteria cells to mucin on the host cells, especially in respiratory infections such as cystic fibrosis ( Figure  1); and having a mechanism to escape the immune system based on the Cell-to-Cell signaling [4,[8][9][10][11][12][13].

Cell-to-cell signaling and immune escape mechanisms
In general, the bacteria control their environmental systems and cell populations through intracellular communications to have the best performance and response according to the demographic and the environmental conditions [14,15]. Pathogenic bacteria take the best advantages of communication capability, as an example, they can overcome the host immune system barrier using the community, in this case, they estimate the cell density using these intercellular signals and given the concentration of signal transmitter factors, and when the density reaches its minimum extent in which the immune system cannot simply cope, bacteria will release the virulence factors, thus, the host immune system will be prevented to deliver rapid responses. The process is controlled by a system in bacteria called "Quorum Sensing" [16][17][18][19][20].

Quorum sensing system in bacteria
Bacteria are known as one of the simplest and the most primitive life forms, which have the single-cell life and their reproduction, feeding and communication mechanisms are identified as basic and simple patterns, however, the complex and interesting mechanisms are provided by studies in recent decades that control bacterial behavior similar to what occurs in multicellular organisms. This mechanism is controlled by quorum sensing system that enables intraspecies and interspecies communication, which involves in terms of biofilm formation, food shortages and environmental stress conditions, such as disinfectants, antibiotics, bacterial colonization, the identification of annoying species, the establishment of normal intestinal flora and the prevention of harmful intestinal flora, etc [21][22][23][24]. An important reason to study this system is to evaluate the mechanisms and to identify the factors affecting on it, in order to inhibit the communication by producing the antagonist of factors and to prevent the biofilm formation, antibiotic resistance spread and chronic and treatmentresistant infections [25][26][27].
As was pointed out, the bacteria often live in the communities, not individual. They can communicate by producing and responding to a series of messages. Quorum sensing is one of the main forms of communication, a two-component term in which quorum means "threshold" and sensing means "feel" that is briefly called QS system. The system is a cell to cell signaling mechanism by which bacteria control the expression of some genes depends on the cell density, a process by which bacteria determine the cell density population and the information is used to regulate the gene expression, so QS system is an example of multicellular behavior in the unicellular world of bacteria [28,29]. The bacteria produce some chemical molecules in the QS phenomenon that are often spreadable and have a low molecular weight that aggregate in the surrounding, these messenger molecules are called "Autoinducer" or "Self-inducer". It is assumed that the balance of chemical messages between organisms and cells is a unique property in eukaryotic cells but recent studies indicate that many bacteria use a group of messenger molecules to coordinate the behaviors. Once the concentration of molecules outside the bacteria exceeds the threshold, the signaling pathways are activated and the bacteria response the messages by altering the gene expression and modulating physiological processes in a collective mode ( Figure 2) [30][31][32].

Quorum sensing in Gram-negative bacteria
Over the past decade, quorum sensing complex have been identified in more than 25 Gram-negative bacteria species. A wide range of major processes in these bacterial species is regulated by quorum sensing, such as bioluminescence, Swarming, Swimming, Twitching, Antibiotics biosynthesis, the growth and spread of biofilm, Conjugation, Sporulation, Virulence markers production, etc [33][34][35][36]. Among these species QS system in Vibrio fischeri, Pseudomonas aeruginosa and Agrobacterium tumefaciens are more considered. However, the studies show the evolution of system and the compliance with the specific requirements of each of the bacteria.
The most studies on quorum sensing are performed on bioluminescence property in Vibrio fisheri. The bacteria lives in symbiosis with eukaryotes, the eukaryotic host provides a nutrient-rich environment for symbiotic bacteria, and in turn, the bacteria produces light that is used to avoid predation and to prey capture. The quorum sensing system in Vibrio fisheri is composed of two regulatory proteins, LuxI and LuxR, which LuxI protein is an autoinducer synthase enzyme and LuxR is a promoter binding protein. Generally, LuxI protein is responsible to biosynthesize a specific messenger molecule called acyl homoserine lactones (AHL/acyl-HSL) or autoinducer. The concentration of autoinducer increases with increasing the cell population. When the concentration reaches a threshold, the autoinducer enters the bacterial cell and binds a protein called R protein. The autoinducer and R protein complex activates the transcription of target gene, and therefore, the bacteria efficiently coordinate the gene expression using quorum sensing system with cell population fluctuations ( Figure 2) [32,37,38]. In recent studies, more special systems than LuxI/R are identified, for example, two pairs of genes in P. aeruginosa (LasI and LasR) have been identified that control the expression of virulence factors. Studies on LuxI and LuxR homologous genes in different bacteria show a similar system and performance (Table 2) [20,39].

Autoinducers
As previously mentioned, autoinducers are small diffusible signaling molecules which are produced by bacteria in the quorum sensing system. These autoinducers (AI) are divided into two categories, autoinducers type-1 or AI-1, which are species-specific and employed for intraspecies communication. Autoinducers type-2 or AI-2 are not specific and act as an international language. Autoinducers type-2 are not only applied for interspecies communication, but also enable the bacteria to communicate microorganisms such as fungi and protozoa to coordinate their own behavior in a population of microorganisms. AI-1 is composed of a acyl homoserine lactone ring (AHL) with an carbon chain which is different in Gram-negative bacteria, therefore, makes the autoinducer thoroughly specific for the species by a series of changes on C3 (locating OXO or Hydroxyl group on it). But, the AI-2 is a furanosyl borate diester. It is noteworthy to mention that the AHL production is only limited to the Proteobacteria of which only 21 species, comprising only 4% of the species, produce AI-2 (Figures 3 and 4) [30,[40][41][42][43][44].

Quorum sensing in Gram-positive bacteria
After changes, the peptides produced in bacterial cells are employed by an ABC channel and secreted into the extracellular space. The induction pathway in the recipient bacterium, called Tow-component signal-transduction, is comprised of two components: Two-component sensor kinase, located in the bacterial cell membrane, acts as a receptor  [45,46].
In the alternative method, the peptide is transferred as a messenger molecule by active transport inside the bacteria using a specific Permease located in the cell membrane, then; the peptide binds a cognate regulatory protein and triggers the regulation of gene expression.
As an example of communication in Gram-positive bacteria, comE/comD system in Streptococcus pneumonia can be mentioned in which the recipient bacterium is potent to receive a foreign DNA. This potential is temporary and occurs only during the logarithmic phase in which the bacteria uptake the DNA regardless its sequence and the species of origin [47][48][49]. The TraR/TraI virulence system in phytopathogen Agrobacterium tumefaciens and hybrid messenger system in Vibrio harveyi are another examples of the role of quorum sensing in pathogenesis. In Agrobacterium tumefaciens Ti plasmid transport is controlled by quorum sensing system [50].

Quorum sensing in Pseudomonas aeruginosa
The quorum sensing system of P. aeruginosa is composed of two pairs of genes as main constitutive genes. One pair consists of LasI and LasR genes, encoding C12-HSL autoinducer synthase and R protein, respectively. Another pair called Rhl is composed of RhlI and RhlR genes, encoding synthase and R protein, respectively ( Figure 5). The synthase enzyme of this pair of QS system produces C4-HSL autoinducer, but produces a small amount of N-hexanoyl-L-homoserine-lactone (HHL) [51]. There is also a regulatory gene named QScR in this system that poduces a regulatory protein which functions as an inhibitor of LasR protein. The functions of the genes of QS system in P. aeruginosa are inter-related and do not operate independently, in other words, it can be said that the expression of Rhl genes is under regulation of Las genes which is performed through the R protein of LasR gene [52].

The main genes that control by QS in P. aeruginosa
Many genes in P. aeruginosa are regulated and expressed by quorum sensing system including pathogenesis genes such as Alkaline protease, Pyocyanin, pyoverdin, cyanide, lipase, twitching movement, alginate, azurin, chitinase, catalase, superoxide dismutase, lasA, lasB, XCP transport machine (a mechanism to deliver bacterial virulence factor), etc [53]. Studies have shown that the Las gene controls the production of virulence factors such as lasB Elastase, lasA Elastase, Alkaline protease and Toxin A. The Rhl gene controls the production of factors such as Rhamnolipids, Elastase, Pyocyanin, Cyanid, moreover, the control can be carried out combined with the Las gene ( Figure 5). The function of Las gene was firstly evaluated by the enzyme activity of lasB elastase, it is why that it was named Las gene in the QS system of P. aeruginosa , and the reason for the naming of Rhl gene is its major role in the Rhamnolipid production. Rhl controls an operon called RhlAB encoding Rhamnosyltransferase enzyme which is essential in the Rhamnolipid production [54].
Regarding the QS system of P. aeruginosa , the followings are noteworthy [32,55,56]: This system is not compatible, meaning that there is no interaction between the products of these two systems; Some bacterial genes are under the control of both pairs of genes, and some others are controlled exclusively by one gene;  These genes are Auto-regulated so that LasR protein/C12-HSL is a positive regulator of the LasI gene and regulates the expression of Rh1R gene, and RhlR protein/C4-HSL regulates the RhlI gene transcription; The R protein of Las system forms a multimer that can bind the Las-Box upstream of the target gene when is bound to the C12-HSL autoinducer. The R protein is a two-domain polypeptide consisting of an N-terminal that binds its autoinducer and a C-terminal that allows the R protein to bind the target gene. In the absence of any autoinducer, N-terminal domain inhibits C-terminal to bind the target gene; The C-terminal domain contains a helix-turn-helix motif by which binds to the Las-Box in 40 bp upstream of the ATG start codon. Las-Box consists of 20 base pair palindrome. The amino acids in the C-terminal domain involve in polymerization; The R protein of Rhl system forms a dimer and binds to the DNA in the presence or absence of C4-HSL, but when it is bound to the autoinducer, it can affect the expression of target genes; It is necessary to have an appropriate concentration of autoinducer in the environment to regulate the genes under the control of QS system, but not sufficient. Simply, the QS-controlled gene expression is not only dependent on the threshold concentration of autoinducer in the environment, but the bacterial growth phase is also important and when the bacteria is not in the appropriate growth condition, the presence of a threshold concentration do not induces the expression of target gene.
As was pointed out, the QS systems play a major role in the regulation of expression of genes by the molecules messengers or autoinducers. Studies have shown that C12-HSL autoinducer causes the IL-8 production in human lung structural cells such as fibroblasts and bronchial epithelial. The autoinducer also stimulates the production of Cyclooxygenase-2 and Prostaglandin E2 in lung fibroblast cells thereby plays a role in inflammation. Moreover, the molecule can induce apoptosis in neutrophils and macrophages. Therefore, the above phenomena lead to the suggestion that the autoinducers besides the regulation of the expression gene producing virulence factors are also a modulator factor for the host immune system function [57,58].
Also, another signal molecule has been discovered in P. aeruginosa called 2-heptyl-3-hydroxyl-4-quinolone. Its structure is not among the homoserine lactone family and is designated Pseudomonas quinolone signal (PQS), which controls the expression of lasB gene beside the Las and Rhl system. The expression of PQS dependents on the LasR and also induces the expression of RhlR gene, therefore, the PQS acts as a link between Las and Rhl systems [59].

Autoinducer synthesis in P. aeruginosa
In this bacterium, the autoinducer is produced by the products of the LasI and RhlI genes which are synthase enzymes. These enzymes utilize S-adenosylmethionine (SAM) and Acyl-acyl carrier protein (Acyl-ACP) substrates to synthesize the autoinducers. The Acyl-ACP is also a mediating factor in fatty acid synthesis process. In the autoinducer synthesis process, SAM binds to the ACP by forming an amide bond between the amino group of homocysteine and the Acyl group of ACP. Subsequently, the autoinducer is produced by the release of Methylthioadenosine and the production of lactone ( Figure 6) [60,61].

The regulation of QS in P. aeruginosa
Considering that the QS system plays an important role in controlling the bacterial genes, therefore, it is important to control and regulate its function. The regulation can be done by the bacteria or by the external factors (Figure 7). This regulation can be done as follows: The regulation of the transcription of LasI and LasR genes that induces changes in the amount of the production of synthase and R protein.
The regulation by the molecular analogs of autoinducers produced by other bacteria or eukaryotic cells.
The regulation by the molecules that can compete with R protein as Agonist or Antagonist.
Enzymatic destruction like AHL degradation enzymes.
In Pseudomonas aeruginosa, salR, vatM, posR, smaR, ponR genes are the suppressor of the expression of LasR and LasI genes in the low density cells.
The inactivation of anti-activator protein QScR encoded by QScR gene is triggered by interacting with LasR protein. In high bacterial cell density, the GacA and PprB genes are also a positive regulators of LasR and Rh1R genes (Table 3) [55,62].
Developing chemical modifications of autoinducers may also be involved in regulating the function of QS system, for instance, the acetylation of homoserine lactone stabilizes the molecular in pH 5 to 6. As was mentioned, the enzyme activities are also involved in regulating QS system, for example, AHL lactonase which was first identified in Variovorax paradoxus helps the organism to utilize AHL as the only source of carbone and energy, consequently causes the AHL degradation, or AHL-acylase in P. aeruginosa by which the bacteria is able to degrade and utilize AHL with long-chains as the only source of carbon and energy, therefore, the enzyme may have a regulatory role. Homoserine lactones are also hydrolased and inactivated through alkalines.
Studies in humans have shown that the epithelial cells of the lung tissue in healthy individuals normally secrete a substance which degrades the autoinducers effective in the qourum sensing process of P. aeruginosa by an enzymatic function and people who are genetically deficient in the production of this inhibitors enzyme shows a higher percentage of cystic fibrosis disease.
The other interesting point is the direct effect of some antibiotics in the regulation of QS system activity, for example, the studies have shown that, in the case of P. aeruginosa , azithromycin antibiotic accelerates the improvement of disease by interfering with the elastase production and the expression of lasR and lasI genes [63].

Quorum sensing as drug target
Due to the spread of antibiotic resistance that today has become one of the main challenges in the field of infectious diseases, therapies avoiding the indiscriminate use of antibiotics are paid special attention. On the other hand, given to the importance of the qourum sensing phenomenon in controlling pathogenicity of bacteria such as P.  aeruginosa , a new generation of antibiotics can be imagined which are designed based on the inhibition of QS system. It is not far from the notion that these inhibitors can be used as a synergism with other medications to reduce their dose. Biofilm that causes resistance against many commonly used antibiotics is regarded as one of the main challenges in treatment. As mentioned, quorum sensing system plays a fundamental role in the regulation, control and formation of biofilm and many virulence factors. Therefore, it is possible that the inhibition of QS regulatory process for removing and reducing the drug resistance in infectious bacteria to be effective.
In 2003, three independent research groups studied the genes under the control of P. aeruginosa QS system, and investigated 388, 315 and 163 genes in the bacterial genome. Studies were performed using the bacteria in which their QS system had been inactivated by mutation and the gene induction was controlled by foreign autoinducers. After studying the gene activity in the presence or absence of autoinducer, 97 genes were reported that jointly are controlled by the QS system (The differences in the results are probably due to the differences in the composition and condition of test medium and ambient oxygen). The studies showed that about 10% of the bacterial genomes are under the control of QS system. Therefore, it was suggested that the bacterial QS system can be considered as a suitable target in order to research about the control of bacterial infection [64][65][66]. This goal can be achieved using several methods including: I) The blockage of R proteins activation by HLA antagonists or HLA antibodies and HLA degredation by chemical or enzymic destruction; II) The metabolism inhibition of AHL by compounds that can compete with substrates which are used in the synthesis of autoinducers; III) Inhibition of regulatory factors that have a positive effect on QS genes; IV) Using antisense oligos against main genes of the QS system [67][68][69].

Quorum sensing signals as biosensor markers
Based on sudies, QS signals can be used as markers for the presence of pathogenic bacteria in clinical and environmental samples by bacterial whole-cell QS biosensors. Generally these biosensors are composed of two main parts: a plasmid construct containing an AHL-responsive transcriptional regulator, relatory promoter, reporter gene; and bacterial host cell ( Figure 8A). However, because QS-deficient mutants can be developed by pathogenic bacteria after colonization in the host, so QS signals should not be employed as the only inputs for microbial biosensors, but can be used for detection of pathogenic bacteria in contaminated environments and products such as groundwater, dairy, and meat products. For example pSB406 and pSB1075 plasmids, and E. coli JM109 as host cell, are QS biosensor system which are used for detection of C4-HSL and C12-HSL autoinducers of P. aeruginosa , respectively. In this system each plasmid is comprised of a R protein producing gene (LasR for pSB1075, RhlR for pSB406) and LuxCDABE operon gene of Vibrio fisheri as a luminescence reporter gene that is activated by specific LasR/RhlR protein-AHL complex ( Figure 8B). It is noteworthy to mention that GRAS hosts like lactic acid bacteria (LABs) (Lactobacillus, Leuconostoc) can be used as carrier host of reporter plasmid for safe applicatios [70][71][72].

Quorum sensing signals and anticancer therapy
The 3-oxo-C12-HSL QS signal of P. aeruginosa inhibits proliferation and induces apoptosis in human breast cancer cell, therefore, it can be considered as an anticancer drug. However this feature may be prevented due to some side effects, for example studies showed that 3-oxo-C12-HSLcan lead to macrophage apoptosis. Nevertheless, this QS signal is a good starting point for developing synthetic AHL homologs with anticancer toxicity and reducing side effects [68,70].
On the other hand, studies in mouse models have shown that some bacteria such as E. coli, Bifidobacterium longum, and attenuated strains of Vibrio cholerae, Salmonella typhimurium, and Listeria monocytogenes have a tendency to localization and prolifration in solid tumors and metastases, perhaps through immune system surveillance, which provides barrier against the host immune system [71,72]. Therefore, bacterial aggregation can be used to produce biosensors that target cancer cells which can be achieved by designing synthetic genetic circuits to recognize cancer microenvironment conditions or cancer cell surface antigenes [73]. Based on this concept, in a study by Anderson et al. a genetic network was developed in E. coli in which the Yersinia pestis invasin gene was regulated by the V. Fischeri LuxI/R system. Followed by high HSL concentrations, invasin will expresse and allows E. coli to bind and invade mammalian cancer-derived cells displaying β1-integrin cell surface receptors. Therefore, whole-cell QS biosensors and gene delivery vehicles can be designed to recognize cancer cell aggregations in vivo [74].

Quorum quenching and biological control
The application of Quorum Quenching (QQ) strategy may be an alternative approach to control bacterial pathogens which employ the Table 3: How the effective genes influence the process of the external regulation of genes in P. aeruginosa QS system [60]. AHL based QS mechanism to regulate pathogenicity. The strategy includes several methods to achieve an artificially increased level of AHLs such as the introduction of a gene coding AHL synthase directly to the plant cells, the employment of AHL-degrading bacteria to protect plants and heterologous expression of genes encoding AHL-degrading enzymes in pathogen cells or in plant tissue. Therefore, bacteria misinterprete the population size and the misinterpretation leads to the production of virulence determinants long before the pathogen population is large enough to sustain infection [75].

RpoS
Several studies showed that the application of bacterial cells producing AHL degrading enzymes can be as biological control agents of plant bacterial diseases. The first example of such purposeful usage for the attenuation of infection symptoms development in plants was transgenic potato and tobacco plants expressing the gene encoding AiiA lactonase manifested strong resistance against infection by P. Carotovorum subsp. carotovorum. Heterologous expression of the aiiA gene encoding the AiiA lactonase from Bacillus sp. in plant pathogens cells P. carotovorum, Burkholderia thailandensis and Erwinia amylovora diseased symptoms development. In order to silence QS system of P. aeruginosa , the same approach was used to reduce 3-oxo-C12-HSL followed by preventing the accumulation C4-HSL. This result was also observed by addition of purified AhlM protein to the growth medium of P. aeruginosa. Consequently, the AHL degrading enzymes together with QS inhibitors may be successfully be applied to disrupt bacterial cell to cell communication and to control bacterial infections [76].

Conclusion
Many bacteria use quorum sensing as a multicellular system to coordinate gene expression according to the density of their local population. By this mechanism bacteria can regulate metabolic, host interactions and environmental processes. So this system can be used as a useful target in medicine and other applications such as the production of biochemicals, microbial biosensors and mixed-species fermentations.