Udaat Mittu

Strength In Numbers - A brief review on quorum sensing Abstract: Considered to be isolated cells once upon a time, it is now almost common knowledge that bacterial cells cooperate with each other via small signalling molecules (autoinducers) which allow them to regulate their physiological processes, a system known as quorum sensing. More than 40 years down the line from the discovery of quorum sensing in Vibrio fischeri and Vibrio harveyi, the perspective on bacteria existing as a community has radically changed. What was merely regarded as a method of ‘auto-induction’ by these bacteria is now known to contribute in almost all aspects of bacterial community living. From horizontal gene transfer events such as conjugation and transformation; to self-defense mechanisms such as antimicrobial production and biofilm formation; all the way upto division events such as sporulation, quorum sensing seems to play a role. For a signalling system which allows both cell to cell communication and intracellular regulation, it seems imperative that we gain better understanding of it to truly comprehend the whole spectrum of variation that exists in these ‘lower’ organisms which seem to be endlessly curious in their ability to function in a manner simpler, yet similar to ‘higher’ organisms. The current review aims then to introduce and briefly explain the major aspects of quorum sensing, how it functions and how it aids in bacterial survival. Introduction: Quorum sensing was discovered when it was observed that Vibrio fischeri and Vibrio harveyi, two luciferase producing bacteria, did not generate any luminescence in the early hours of incubation as a pure culture, however, as the concentration of the cells increased, the luminosity of the culture increased with it. One possibility behind this observation was thought to be that the individual luminosity of the cell could be low, thus, luminosity is only observable at high concentrations, or as we know now to be true, it could be because of the ability of the individual bacterium in the culture to assess the population density around it and as a result generate luciferase. In the end, high amount of autoinducer levels were detected when the cultures were at a high enough cell density and thus the idea of cell to cell communication between bacteria was born. (1) Since then, such cellcell communication has been explored in bacteria and multiple types of systems have been detected, but in general, they can be broken down into two kinds of systems; first as observed in gram negative bacteria where there is a dedicated autoinducer sensing protein which in turn regulates gene expression and second as seen in gram positive bacteria, where a prototypical two component system (TCS) serves the sensory function of the system. Beyond this, some systems can be a hybrid of the two aforementioned ones as is the case for Vibrio harveyi. (2,3) Beyond all these variations, what remains constant in every such system are the following two underlying processes: 1. Synthesis of autoinducers and secretion of autoinducers into the medium 2. Detection of autoinducers via binding of sensory proteins to their cognate autoinducer when the said inducer is present at a threshold concentration. (4) Thus, one must breakdown these processes in order to have a proper understanding of how the system works. Synthesis of autoinducers: Small diffusible signalling molecules, called autoinducers, serve as the method of communication in bacterial communities. N-acyl homoserine lactones (AHLs; gram negative bacteria) or autoinducing peptides (AIPs; gram positive bacteria) are secreted by cells and serve as a linch pin for the functioning of the system. (2) AHLs are generated by dedicated LuxI family proteins (named after LuxI of Vibrio fischeri) such as TraI in Agrobacterium tumefaciens. The mechanism of production of AHLs uses Sadenosylmethionine (SAM) and acyl-acyl carrier proteins (ACPs) as the substrates for the reaction. TraI serves as a catalyst which aids in the formation of an amide linkage between SAM and ACPs. Release of methylioadenosine occurs following lactonization of this SAM-ACP complex. The resultant molecule is N-(3-oxooctanoyl)-homoserine lactone. This resultant molecule is then released into the medium where it serves its signalling function. It is important to note that even though AHLs are generated by a family of proteins, AHLs and their sensors are not mutually exchangeable. (5) The mechanism for the reaction is drawn in the figure-1. In contrast, gram positive bacteria employ small peptides for the same function. These small peptides are transported out of the cell using ABC (ATP binding cassette) transporters. (6) Figure-1: Mechanism of generation of N-(3-oxooctanoyl)homoserine by TraI of Agrobacterium tumefaciens. fi Reprinted from Moré, Margret et al Enzymatic synthesis of a quorum-sensing autoinducer through use of de ned substrates (5) Once these autoinducer molecules are released into the medium, they can be detected by dedicated detectors present in bacteria in the vicinity which sets off various physiological processes in the recipient. The next section thus explores the other side of the fence. Detection of autoinducers: Once autoinducers are released in the medium, it is upto individual bacteria to detect these molecules. 3 kinds of systems exist in this classification criterion: 1. Systems with dedicated sensors (AHL sensors) 2. Systems using TCSs for sensing (Peptide sensors) 3. Hybrid systems which use both of the above methods. Let’s follow an example of each of these systems and see how sensing functions in these systems. I: LuxI/LuxR system of Vibrio fischeri (AHL based): Vibrio fischeri was one of the organisms in which quorum sensing was originally discovered, it follows then that a large amount of knowledge has been accumulated about how exactly the system functions in this bug. The synthesis of AHL by LuxI occurs in a manner similar to what has been discussed in the previous section. Many synthesis proteins of quorum sensing responses exist, such as LasI and RhlI of Pseudomonas aeruginosa and ExpI and CarI of Erwinia carotovora, all of which are considered members of the LuxI family of proteins. (2,7,8,9) Following that, the sensing is handled by proteins similar to LuxR LuxR proteins have two functions: sensing AHLs and regulating the downstream response after binding to their dedicated signalling molecules. These distinct functions are neatly distributed across the amino-terminal and carboxyl-terminal domains of the protein. AHLs bind to the aminoterminal domain, whereas the carboxyl-terminal domain harbours DNA binding helix-turn-helix motif. The protein functions as follows - when the AHL is not bound, the amino-terminal domain blocks the carboxyl-terminal domain, as a result, DNA binding motif is masked. Once binding of AHLs to the amino-terminus of occurs, the block is removed from the carboxyl-terminus. This opens up specific residues in the protein which allow for multiple protein units to multimerize. It is in this multimer state that LuxR can bind to DNA and thus regulate various genes in the bacterium’s genome via direct binding. LuxR usually binds to a palindromic ≅20bp region which is present 40bp upstream of the transcription start site of the gene. This palindromic region is therefore aptly named the “lux box”. These lux box regions seem to be mutually exchangeable between multiple members of the LuxI/LuxR family of proteins as has been seen using transgenics experiments. (10) II: ComP/ComA system of Bacillus subtilis: ComP/ComA system controls the choice between sporulation and competence in Bacillus subtilis. 2 peptides, ComX (10 amino acids) and CSF (55 amino acids), are the signalling molecules of choice for this system. These peptides are expressed from the comX gene. These molecules are released in the medium and interact with the ComP/ComA TCS of the bug. (11) A TCS consists of two components - a sensory kinase and a response regulator. When a signalling molecule attaches to the sensory kinase, it gets phosphorylated. This kinase in turn phosphorylates the response regulator which can then bind to various target genomic regions. Thus, such a phospho-cascade mechanism allows the TCS to perform its function. ComP/ComA TCS performs this function for both autoinducers of Bacillus subtilis. The network is a bit more complicated with a lot of crosstalk between various members. (11) Overall, CSF levels dictate the decision between sporulation vs competence, with lower levels promoting the latter. Let’s look at both of these autoinducers individually. 1. ComX - ComX binds to ComP sensory kinase, which downstream phosphorylates ComA. PComA causes the expression of ComS, which protects a protein called ComK, a transcriptional activator, from proteolytic degradation. This ComK functions as a response regulator and binds to various components of the genome and drives the bug towards competence. (12) 2. CSF - CSF is a 55 amino acid pentapeptide. CSF present in the medium is imported via an ABC transporter. At low levels, CSF binds to RapC which is an interacting partner of P-ComA. This increases the levels of free P-ComA and drives the cell towards a competence fate. However, at high concentrations, CSF inhibits the expression of comS, resulting in the degradation of ComK and thus, competence is avoided. CSF also binds to RapB. RapB is a phosphatase which during normal cell function acts as an inhibitor of Spo0A by dephosphorylating it. But, when RapB is quenched by CSF, Spo0A escapes that fate and thus, sporulation is promoted. (13,14) This network has also been represented diagrammatically in figure-2. Figure-2 Competence vs Sporulation in Bacillus subtilis. In the CSF pathway, dotted lines indicate conditions where CSF levels are low, solid lines indicate high CSF levels. Image generated using biorender.com III: Hybrid network of Vibrio harveyi: In Vibrio fischeri, both sides of the coin exist. A sensory protein like gram negative bugs and a TCS like gram positive bugs. For each of the two systems, it has a different autoinducer. N-(3hydroxybutanoyl)-homoserine lactone serves as AI-1. It is generated by LuxLM proteins, which share no homology with LuxI. It remains unknown what the exact mechanism of the reaction is, however, it is theorised that the mechanism must be similar to that of LuxI protein family. The other autoinducer, AI-2, is an unknown autoinducer generated by LuxS. Both of the autoinducers are detected by their own dedicated hybrid TCS. These TCSs are hybrids which contain both the sensory kinase and response regulator functions in different domains of the same molecule. LuxN detects AI-1 and LuxQ handles AI-2. Both LuxL and LuxN transfer a phosphate group to LuxU which relays that phosphate to LuxO. P-LuxO is the one which finally binds to the genomic regions and prevents the activation of the luxCDABE operon via an unknown inhibitor. (15,16) When the medium has low cell density, the response regulators autophosphorylate which downstream inhibits the expression of luciferase from luxCDABE. When the cell density increases however, the enrichment of AI-1 and AI-2 increases. Once these bind to their dedicated TCS, instead of acting as a kinase, these TCS proteins act as a phosphatase instead, and thus reduce the amount of phosphate being relayed to LuxO. This leads to inhibition of the inhibitor of luxCDABE, as a result of which luciferase is generated and bioluminescence is observed. (17,18) The mechanism has been diagrammatically represented in figure-3. Figure-3: Hybrid network of Vibrio harveyi Conclusion: In general, quorum sensing mechanisms are generally one of the three aforementioned types. These mechanisms use signalling molecules to enable communication in bacteria, generating a community of sorts. It must be noted that besides the general types mentioned here, other kinds of quorum sensing systems may also exist. An example of one would be the system of Myxococcus xanthus, in which the autoinducers are generated outside the cells by the action of extracellular proteases. The snipped peptides combine to form a single signal for a TCS and affect spore formation in this bug. (19) As mentioned in the review already, autoinducers and their sensors, although generated by similar proteins, may not be mutually exchangeable as seen in LuxI/LuxR homologs. Mild changes in the autoinducer for a dedicated sensor may cause the downstream effect to be greatly altered. Such alterations are especially observable for acyl group modifications of AHLs. (20) It would be pertinent to mention here that quorum sensing may not be limited to just bacteria. In a recent article by Erez et al in Nature in 2017, it has been shown that bacteriophages of the SPbeta group which infect Bacillus species generate small 6 amino acid peptides which they use to communicate among each other. High concentrations of the peptide cause the phages to choose lysogeny over lysis. Thus, quorum sensing contributes to cell fate decisions in some viruses as well. (21) In light of this, it might be worth exploring more avenues when it comes to quorum sensing beyond just bacteria. Beyond just looking at the systems themselves, one can also look at possible disruptors of these systems. As happens during the course of evolution, natural answers against harmful organisms are developed. Since quorum sensing allows bacteria to adjust to a large variation of niches, it is obvious that higher organisms which are infected by such bugs have developed inhibitors which disrupt this cell-cell communication. Furanones generated by some seaweeds have been shown to be natural inhibitors of quorum sensing systems. These natural counters may help us combat harmful pathogens down the line. (22) In the end, our understanding of quorum sensing needs to increase at a rapid pace. With antimicrobial resistant ESKAPE pathogens peeking over the horizon, any knowledge of how bacteria live through adverse conditions, how they adapt and communicate in order to proliferate effectively might prove endlessly useful. Towards that endeavour, more and more inquiries of variations in such systems, their plasticity and rigidity and finally, the conditions in which they fail need to be explored. Beyond all that, it is fascinating how microscopic creatures, born millions of years ago developed an elementary communication network in order to assist one another. Thus, the academic allure of this topic could be another bait that seems luscious enough to bite on for scientists across the globe. References: 1. Nealson’, K. H., & Hastings2, J. W. (1979). Bacterial bioluminescence: its control and ecological significance. Microbiological Reviews, 43(4), 496–518. https://doi.org/10.1128/MR- 2. M. B. M., & Bassler, B. L. (2003). Quorum Sensing in Bacteria. http://Dx.Doi.Org/10.1146/ Annurev.Micro.55.1.165, 12, 48. https://doi.org/10.1146/ANNUREV.MICRO.55.1.165 3. Bassler, B. L., Wright, M., & Silverman, M. R. (1994). Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway. Molecular Microbiology, 13(2), 273–286. https://doi.org/10.1111/J-.TB00422.X 4. Pan, J., & Ren, D. (2009). Quorum sensing inhibitors: a patent overview. Http://Dx.Doi.Org/ 10.1517/-, 19(11),-. https://doi.org/10.1517/- 5. Moré, M. I., Finger, L. D., Stryker, J. L., Fuqua, C., Eberhard, A., & Winans, S. C. (1996). Enzymatic synthesis of a quorum-sensing autoinducer through use of defined substrates. Science,-),-. https:// doi.org/10.1126/SCIENCE- 6. Microbiology, B. B.-C. opinion in, & 1999, undefined. (n.d.). How bacteria talk to each other: regulation of gene expression by quorum sensing. Elsevier. Retrieved December 15, 2021, from https:// www.sciencedirect.com/science/article/pii/S- 7. Brint, J. M., & Ohman, D. E. (1995). Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR-RhlI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive LuxR-LuxI family. Journal of Bacteriology, 177(24),-. https://doi.org/10.1128/ JB- 8. Passador, L., Cook, J., Gambello, M., … L. R.-, & 1993, undefined. (n.d.). Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science.Sciencemag.Org. Retrieved December 15, 2021, from https://science.sciencemag.org/content/260/5111/1127.abstract 9. Jones, S., Yu, B., Bainton, N. J., Birdsall, M., Bycroft, B. W., Chhabra, S. R., Cox, A. J. R., Golby, P., Reeves, P. J., Stephens, S., Winson, M. K., Salmond, G. P. C., Stewart, G. S. A. B., & Williams, P. (1993). The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa. EMBO Journal, 12(6),-. https://doi.org/10.1002/ J-.TB05902.X 10. Stevens, A. M., & Greenberg, E. P. (1997). Quorum sensing in Vibrio fischeri: Essential elements for activation of the luminescence genes. Journal of Bacteriology, 179(2), 557–562. https://doi.org/10.1128/ JB-. Grossman, A. D. (1995). Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis. Annual Review of Genetics, 29, 477–508. https://doi.org/10.1146/ ANNUREV.GE-. Turgay, K., Hamoen, L. W., Venema, G., & Dubnau, D. (n.d.). Biochemical characterization of a molecular switch involving the heat shock protein ClpC, which controls the activity of ComK, the competence transcription factor of. Genesdev.Cshlp.Org. Retrieved December 15, 2021, from http://genesdev.cshlp.org/content/ 11/1/119.short 13. Solomon, J. M., Lazazzera, B. A., & Grossman, A. D. (1996). Purification and characterization of an extracellular peptide factor that affects two different developmental pathways in Bacillus subtilis. Genesdev.Cshlp.Org. http://genesdev.cshlp.org/content/10/16/2014.short 14. M. P.-P. of the N. A., & 1997, undefined. (1997). A peptide export–import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay. National Acad Sciences, 94,-. https://www.pnas.org/content/94/16/8612.short 15. Freeman, J. A., & Bassler, B. L. (1999). A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi. Molecular Microbiology, 31(2), 665–677. https:// doi.org/10.1046/J-.X 16. Freeman, J. A., & Bassler, B. L. (1999). Sequence and function of LuxU: A two-component phosphorelay protein that regulates quorum sensing in Vibrio harveyi. Journal of Bacteriology, 181(3), 899–906. https://doi.org/ 10.1128/JB-. Martin, M., Showalter, R., & Silverman, M. (1989). Identification of a locus controlling expression of luminescence genes in Vibrio harveyi. Journal of Bacteriology, 171(5),-. https://doi.org/10.1128/ JB-. Lilley, B. N., & Bassler, B. L. (2000). Regulation of quorum sensing in Vibrio harveyi by LuxO and Sigma-54. Molecular Microbiology, 36(4), 940–954. https://doi.org/10.1046/J-.X 19. Dworkin, M., Science, D. K.-, & 1985, undefined. (n.d.). Cell interactions in myxobacterial growth and development. Science.Sciencemag.Org. Retrieved December 15, 2021, from https:// science.sciencemag.org/content/230/4721/18.abstract 20. Gray, K. M., Passador, L., Iglewski, B. H., & Greenberg, E. P. (1994). Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa. Journal of Bacteriology, 176(10),-. https://doi.org/10.1128/JB-. Erez, Z., Steinberger-Levy, I., Shamir, M., Doron, S., Stokar-Avihail, A., Peleg, Y., Melamed, S., Leavitt, A., Savidor, A., Albeck, S., Amitai, G., & Sorek, R. (2017). Communication between viruses guides lysis– lysogeny decisions. Nature-:7638,-), 488–493. https://doi.org/10.1038/nature-. Manefield, M., De Nys, R., Kumar, N., Read, R., Givskov, M., Steinberg, P., & Kjelleberg, S. (1999). Evidence that halogenated furanones from Delisea pulchra inhibit acylated homoserine lactone (AHL)-mediated gene expression by displacing the AHL signal from its receptor protein. Microbiology, 145(2), 283–291. https:// doi.org/10.1099/-