Quorum sensing

For the bacteria to use quorum sensing constitutively, they must possess three abilities: secretion of a signaling molecule, secretion of an autoinducer (to detect the change in concentration of signaling molecules), and regulation of gene transcription as a response.[9] This process is highly dependent on the diffusion mechanism of the signaling molecules. QS signaling molecules are usually secreted at a low level by individual bacteria. At low cell density, the molecules may just diffuse away. At high cell density, the local concentration of signaling molecules may exceed its threshold level, and trigger changes in gene expression.[11]

Gram-positive bacteria use autoinducing peptides (AIP) as their autoinducers.[12]

When gram-positive bacteria detect high concentration of AIPs in their environment, that happens by way of AIPs binding to a receptor to activate a kinase. The kinase phosphorylates a transcription factor, which regulates gene transcription. This is called a two-component system.

Another possible mechanism is that AIP is transported into the cytosol, and binds directly to a transcription factor to initiate or inhibit transcription.[12]

Gram-negative bacteria produce N-acyl homoserine lactones (AHL) as their signaling molecule.[12] Usually AHLs do not need additional processing, and bind directly to transcription factors to regulate gene expression.[11]

Some gram-negative bacteria may use the two-component system as well.[12]

The bioluminescent bacterium A. fischeri is the first organism in which QS was observed. It lives as a mutualistic symbiont in the photophore (or light-producing organ) of the Hawaiian bobtail squid. When A. fischeri cells are free-living (or planktonic), the autoinducer is at low concentration, and, thus, cells do not show luminescence. However, when the population reaches the threshold in the photophore (about 10¹¹ cells/ml), transcription of luciferase is induced, leading to bioluminescence. In A. fischeri bioluminescence is regulated by AHLs (N-acyl-homoserine lactones) which is a product of the LuxI gene whose transcription is regulated by the LuxR activator. LuxR works only when AHLs binds to the LuxR.

Curvibacter sp. is a gram-negative curved rod-formed bacterium which is the main colonizer of the epithelial cell surfaces of the early branching metazoan Hydra vulgaris.[13][14] Sequencing the complete genome uncovered a circular chromosome (4.37 Mb), a plasmid (16.5 kb), and two operons coding each for an AHL (N-acyl-homoserine lactone) synthase (curI1 and curI2) and an AHL receptor (curR1 and curR2).[14] Moreover, a study showed that these host associated Curvibacter bacteria produce a broad spectrum of AHL, explaining the presence of those operons.[14] As mentioned before, AHL are the quorum sensing molecules of gram-negative bacteria, which means Curvibacter has a quorum sensing activity.

Even though their function in host-microbe interaction is largely unknown, Curvibacter quorum-sensing signals are relevant for host-microbe interactions.[14] Indeed, due to the oxidoreductase activity of Hydra, there is a modification of AHL signalling molecules (3-oxo-homoserine lactone into 3-hydroxy-homoserine lactone) which leads to a different host-microbe interaction. On one hand, a phenotypic switch of the colonizer Curvibacter takes place. The most likely explanation is that the binding of 3-oxo-HSL and 3-hydroxy-HSL causes different conformational changes in the AHL receptors curR1 and curR2. As a result, there is a different DNA-binding motif affinity and thereby different target genes are activated.[14] On the other hand, this switch modifies its ability to colonize the epithelial cell surfaces of Hydra vulgaris.[14] Indeed, one explanation is that with a 3-oxo-HSL quorum-sensing signal, there is an up-regulation of flagellar assembly. Yet, flagellin, the main protein component of flagella, can act as an immunomodulator and activate the innate immune response in Hydra. Therefore, bacteria have less chance to evade the immune system and to colonize host tissues.[14] Another explanation is that 3-hydroxy-HSL induces carbon metabolism and fatty acid degradation genes in Hydra. This allows the bacterial metabolism to adjust itself to the host growth conditions, which is essential for the colonization of the ectodermal mucus layer of Hydrae.[14]

In the gram-negative bacterium Escherichia coli (E. coli), cell division may be partially regulated by AI-2-mediated quorum sensing. This species uses AI-2, which is produced and processed by the lsr operon. Part of it encodes an ABC transporter, which imports AI-2 into the cells during the early stationary (latent) phase of growth. AI-2 is then phosphorylated by the LsrK kinase, and the newly produced phospho-AI-2 can be either internalized or used to suppress LsrR, a repressor of the lsr operon (thereby activating the operon). Transcription of the lsr operon is also thought to be inhibited by dihydroxyacetone phosphate (DHAP) through its competitive binding to LsrR. Glyceraldehyde 3-phosphate has also been shown to inhibit the lsr operon through cAMP-CAPK-mediated inhibition. This explains why, when grown with glucose, E. coli will lose the ability to internalize AI-2 (because of catabolite repression). When grown normally, AI-2 presence is transient.

E. coli and Salmonella enterica do not produce AHL signals commonly found in other gram-negative bacteria. However, they have a receptor that detects AHLs from other bacteria and change their gene expression in accordance with the presence of other "quorate" populations of gram-negative bacteria.[15]

Salmonella encodes a LuxR homolog, SdiA, but does not encode an AHL synthase. SdiA detects AHLs produced by other species of bacteria including Aeromonas hydrophila, Hafnia alvei, and Yersinia enterocolitica.[16] When AHL is detected, SdiA regulates the rck operon on the Salmonella virulence plasmid (pefI-srgD-srgA-srgB-rck-srgC) and a single gene horizontal acquisition in the chromosome srgE.[17][18] Salmonella does not detect AHL when passing through the gastrointestinal tracts of several animal species, suggesting that the normal microbiota does not produce AHLs. However, SdiA does become activated when Salmonella transits through turtles colonized with Aeromonas hydrophila or mice infected with Yersinia enterocolitica.[19][20] Therefore, Salmonella appears to use SdiA to detect the AHL production of other pathogens rather than the normal gut flora.

The opportunistic pathogen Pseudomonas aeruginosa uses quorum sensing to coordinate the formation of biofilm, swarming motility, exopolysaccharide production, virulence, and cell aggregation.[21] These bacteria can grow within a host without harming it until they reach a threshold concentration. Then they become aggressive, developing to the point at which their numbers are sufficient to overcome the host's immune system, and form a biofilm, leading to disease within the host as the biofilm is a protective layer encasing the bacterial population. Another form of gene regulation that allows the bacteria to rapidly adapt to surrounding changes is through environmental signaling. Recent studies have discovered that anaerobiosis can significantly impact the major regulatory circuit of quorum sensing. This important link between quorum sensing and anaerobiosis has a significant impact on the production of virulence factors of this organism.[22] There is hope among some humans that the therapeutic enzymatic degradation of the signaling molecules will be possible when treating illness caused by biofilms, and prevent the formation of such biofilms and possibly weaken established biofilms. Disrupting the signaling process in this way is called quorum sensing inhibition.

It has recently been found that Acinetobacter sp. also show quorum sensing activity. This bacterium, an emerging pathogen, produces AHLs.[23] Acinetobacter sp. shows both quorum sensing and quorum quenching activity. It produces AHLs and can also degrade the AHL molecules.[23]

This bacterium was previously considered a fish pathogen, but it has recently emerged as a human pathogen.[24] Aeromonas sp. have been isolated from various infected sites from patients (bile, blood, peritoneal fluid, pus, stool and urine). All isolates produced the two principal AHLs, N-butanoylhomoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL). It has been documented that Aeromonas sobria has produced C6-HSL and two additional AHLs with N-acyl side chain longer than C6.[25]

The YenR and YenI proteins produced by the gammaproteobacterium Yersinia enterocolitica are similar to Aliivibrio fischeri LuxR and LuxI.[26][27] YenR activates the expression of a small non-coding RNA, YenS. YenS inhibits YenI expression and acylhomoserine lactone production.[28] YenR/YenI/YenS are involved in the control of swimming and swarming motility.[27][28]

Three-dimensional structures of proteins involved in quorum sensing were first published in 2001, when the crystal structures of three LuxS orthologs were determined by X-ray crystallography.[29] In 2002, the crystal structure of the receptor LuxP of Vibrio harveyi with its inducer AI-2 (which is one of the few biomolecules containing boron) bound to it was also determined.[30] Many bacterial species, including E. coli, an enteric bacterium and model organism for gram-negative bacteria, produce AI-2. A comparative genomic and phylogenetic analysis of 138 genomes of bacteria, archaea, and eukaryotes found that "the LuxS enzyme required for AI-2 synthesis is widespread in bacteria, while the periplasmic binding protein LuxP is present only in Vibrio strains," leading to the conclusion that either "other organisms may use components different from the AI-2 signal transduction system of Vibrio strains to sense the signal of AI-2 or they do not have such a quorum sensing system at all."[31] Vibrio species utilize Qrr RNAs, small non-coding RNAs, that are activated by these autoinducers to target cell density master regulators. Farnesol is used by the fungus Candida albicans as a quorum sensing molecule that inhibits filamentation.[32]

A database of quorum-sensing peptides is available under the name Quorumpeps.[33][34]

Certain bacteria can produce enzymes called lactonases that can target and inactivate AHLs. Researchers have developed novel molecules which block the signalling receptors of bacteria ("Quorum quenching"). mBTL is a compound that has been shown to inhibit quorum sensing and decrease the amount of cell death by a significant amount.[35] Additionally, researchers are also examining the role of natural compounds (such as caffeine) as potential quorum sensing inhibitors.[36] Research in this area has been promising and could lead to the development of natural compounds as effective therapeutics.

The majority of quorum sensing systems that fall under the "two-gene" (an autoinducer synthase coupled with a receptor molecule) paradigm as defined by the Vibrio fischeri system occur in the gram-negative Pseudomonadota. A comparison between the Pseudomonadota phylogeny as generated by 16S ribosomal RNA sequences and phylogenies of LuxI-, LuxR-, or LuxS-homologs shows a notably high level of global similarity. Overall, the quorum sensing genes seem to have diverged along with the Pseudomonadota phylum as a whole. This indicates that these quorum sensing systems are quite ancient, and arose very early in the Pseudomonadota lineage.[37][38]

Although examples of horizontal gene transfer are apparent in LuxI, LuxR, and LuxS phylogenies, they are relatively rare. This result is in line with the observation that quorum sensing genes tend to control the expression of a wide array of genes scattered throughout the bacterial chromosome. A recent acquisition by horizontal gene transfer would be unlikely to have integrated itself to this degree. Given that the majority of autoinducer–synthase/receptor pairs occur in tandem in bacterial genomes, it is also rare that they switch partners and so pairs tend to co-evolve.[38]

In quorum sensing genes of Gammaproteobacteria, which includes Pseudomonas aeruginosa and Escherichia coli, the LuxI/LuxR genes form a functional pair, with LuxI as the auto-inducer synthase and LuxR as the receptor. Gammaproteobacteria are unique in possessing quorum sensing genes, which, although functionally similar to the LuxI/LuxR genes, have a markedly divergent sequence.[38] This family of quorum-sensing homologs may have arisen in the Gammaproteobacteria ancestor, although the cause of their extreme sequence divergence yet maintenance of functional similarity has yet to be explained. In addition, species that employ multiple discrete quorum sensing systems are almost all members of the Gammaproteobacteria, and evidence of horizontal transfer of quorum sensing genes is most evident in this class.[37][38]

Next to the potential antimicrobial functionality, quorum-sensing derived molecules, especially the peptides, are being investigated for their use in other therapeutic domains as well, including immunology, central nervous system disorders and oncology. Quorum-sensing peptides have been demonstrated to interact with cancer cells, as well as to permeate the blood–brain barrier reaching the brain parenchyma.[39][40][41]

When aggregated in high enough densities, some bacteria may form biofilms to protect themselves from biotic or abiotic threats. Biofilms may also serve to transport nutrients into the microbial community or transport toxins out by means of channels that permeate the extracellular polymeric matrix (like cellulose) that holds the cells together. Finally, biofilms are an ideal environment for horizontal gene transfer through either conjugation or environmental DNA (eDNA) that exists in the biofilm matrix.[42]

The process of biofilm development is often triggered by environmental signals, and bacteria are proven to require flagella to successfully approach a surface, adhere to it, and form the biofilm.[42] As cells either replicate or aggregate in a location, the concentration of autoinducers outside of the cells increases until a critical mass threshold is reached. At this point, it is energetically unfavorable for intracellular autoinducers to leave the cell and they bind to receptors and trigger a signaling cascade to initiate gene expression and begin secreting an extracellular polysaccharide to encase themselves inside.[43]

Methanosaeta harundinacea 6Ac, a methanogenic archaeon, produces carboxylated acyl homoserine lactone compounds that facilitate the transition from growth as short cells to growth as filaments.[44]

Closantel and triclosan are known inhibitors of quorum sensing enzymes.[58] Closantel induces aggregation of the histidine kinase sensor in two-component signalling. The latter disrupts the synthesis of a class of signalling molecules known as N-acyl homoserine lactones (AHLs) by blocking the enoyl-acyl carrier protein (ACP) reductase.[58][59]

Two groups of well-known mimicking molecules include halogenated furanones, which mimic AHL molecules, and synthetic Al peptides (AIPs), which mimic naturally occurring AIPs. These groups inhibit receptors from binding substrate or decrease the concentration of receptors in the cell.[58] Furanones have also been found to act on AHL-dependant transcriptional activity, whereby the half life of the autoinducer-binding LuxR protein is significantly shortened.[60]

Recently, a well-studied quorum quenching bacterial strain (KM1S) was isolated and its AHL degradation kinetics were studied using rapid resolution liquid chromatography (RRLC).[61] RRLC efficiently separates components of a mixture to a high degree of sensitivity, based on their affinities for different liquid phases.[62] It was found that the genome of this strain encoded an inactivation enzyme with distinct motifs targeting the degradation of AHLs.[61]

As mentioned before, N-acyl-homoserine lactones (AHL) are the quorum sensing signaling molecules of the gram-negative bacteria. However, these molecules may have different functional groups on their acyl chain, and also a different length of acyl chain. Therefore, there exist many different AHL signaling molecules, for example, 3-oxododecanoyl-L-homoserine lactone (3OC12-HSL) or 3-hydroxydodecanoyl-L-homoserine lactone (3OHC12-HSL). The modification of those quorum sensing (QS) signaling molecules is another sort of quorum quenching. This can be carried out by an oxidoreductase activity.[14] As an example, we will discuss the interaction between a host, Hydra vulgaris, and the main colonizer of its epithelial cell surfaces, Curvibacter spp. Those bacteria produce 3-oxo-HSL quorum sensing molecules.[14] However, the oxidoreductase activity of the polyp Hydra is able to modify the 3-oxo-HSL into their 3-hydroxy-HSL counterparts.[14] We can characterize this as quorum quenching since there is an interference with quorum sensing molecules. In this case, the outcomes differ from simple QS inactivation: the host modification results in a phenotypic switch of Curvibacter, which modifies its ability to colonize the epithelial cell surfaces of H. vulgaris.[14]

Applications of quorum quenching that have been exploited by humans include the use of AHL-degrading bacteria in aquacultures to limit the spread of diseases in aquatic populations of fish, mollusks and crustaceans.[63] This technique has also been translated to agriculture, to restrict the spread of pathogenic bacteria that use quorum sensing in plants.[63][64] Anti-biofouling is another process that exploits quorum quenching bacteria to mediate the dissociation of unwanted biofilms aggregating on wet surfaces, such as medical devices, transportation infrastructure and water systems.[63][65] Quorum quenching is recently studied for the control of fouling and emerging contaminants in electro membrane bioreactors (eMBRs) for the advanced treatment of wastewater.[66] Extracts of several traditional medicinal herbs display quorum quenching acivity, and have potential antibacterial applications.[67][68]

Colonies of the ant Temnothorax albipennis nest in small crevices between rocks. When the rocks shift and the nest is broken up, these ants must quickly choose a new nest to move into. During the first phase of the decision-making process, a small portion of the workers leave the destroyed nest and search for new crevices. When one of these scout ants finds a potential nest, she assesses the quality of the crevice based on a variety of factors including the size of the interior, the number of openings (based on light level), and the presence or absence of dead ants.[69][70] The worker then returns to the destroyed nest, where she waits for a short period before recruiting other workers to follow her to the nest that she has found, using a process called tandem running. The waiting period is inversely related to the quality of the site; for instance, a worker that has found a poor site will wait longer than a worker that encountered a good site.[71] As the new recruits visit the potential nest site and make their own assessment of its quality, the number of ants visiting the crevice increases. During this stage, ants may be visiting many different potential nests. However, because of the differences in the waiting period, the number of ants in the best nest will tend to increase at the greatest rate. Eventually, the ants in this nest will sense that the rate at which they encounter other ants has exceeded a particular threshold, indicating that the quorum number has been reached.[72] Once the ants sense a quorum, they return to the destroyed nest and begin rapidly carrying the brood, queen, and fellow workers to the new nest. Scouts that are still tandem-running to other potential sites are also recruited to the new nest, and the entire colony moves. Thus, although no single worker may have visited and compared all of the available options, quorum sensing enables the colony as a whole to quickly make good decisions about where to move.

Honey bees (Apis mellifera) also use quorum sensing to make decisions about new nest sites. Large colonies reproduce through a process called swarming, in which the queen leaves the hive with a portion of the workers to form a new nest elsewhere. After leaving the nest, the workers form a swarm that hangs from a branch or overhanging structure. This swarm persists during the decision-making phase until a new nest site is chosen.

The quorum sensing process in honey bees is similar to the method used by Temnothorax ants in several ways. A small portion of the workers leave the swarm to search out new nest sites, and each worker assesses the quality of the cavity it finds. The worker then returns to the swarm and recruits other workers to her cavity using the honey bee waggle dance. However, instead of using a time delay, the number of dance repetitions the worker performs is dependent on the quality of the site. Workers that found poor nests stop dancing sooner, and can, therefore, be recruited to the better sites. Once the visitors to a new site sense that a quorum number (usually 10–20 bees) has been reached, they return to the swarm and begin using a new recruitment method called piping. This vibration signal causes the swarm to take off and fly to the new nest location. In an experimental test, this decision-making process enabled honey bee swarms to choose the best nest site in four out of five trials.[73][74]