Team:TU Darmstadt/sensing-background

Sensing – Background – TUDA iGEM 2021

Sensing – Background

Background of quorum sensing and acyl homoserine lactones

Explanation of the sensing circuit in E. coli and in B. subtilis

Collected results and discussion of our laboratory work

Quorum Sensing

Quorum sensing (QS) is a mechanism which allows bacteria to communicate via chemical signaling molecules called autoinducers. It can take place within and between species. By sensing autoinducers, the bacteria are capable to regulate gene expression depending on the cell population density (Figure 1). This can affect physiological activities like virulence, competence or biofilm formation.​1​

Figure 1. Schematic overview of quorum sensing (QS). Bacteria like B. subtilis or P. aeruginosa can release and sense autoinducers, i.e. acyl homoserine lactones (AHL) in case of P. aeruginosa, to regulate their gene expression.

Bacteria can be divided into two different groups based on the composition of the cell wall. Gram-positive bacteria possess a thick cell wall with a high proportion of peptidoglycan. Gram-negative bacteria possess a thin cell wall with an additional outer membrane (Figure 2).​2​ Bacteria also vary in their quorum sensing regarding their signaling molecule.

Figure 2. Illustration of Gram-positive (left) and Gram-negative (right) cell walls. Visualization on the difference between Gram-positive and Gram-negative cell wall relating to the thickness of peptidoglycan layers.

Gram-positive bacteria, like our Bacillus subtilis sleeper cells commonly use oligopeptides as autoinducers. To detect the autoinducer the cells need a two-component system. First, the peptide binds to a membrane receptor. Second, a kinase is activated and phosphorylates a transcription factor. This factor then regulates gene expression by binding to its associated promoter region. We use this two-component system to create a kill-switch for our B. subtilis cells, preventing them to survive when leaving the biofilm.

Gram-negative bacteria use the LuxI/LuxR- quorum sensing system. For that, acyl homeserine lactones (AHL) are produced as autoinducers by LuxI-like proteins. In contrast to Gram-positive bacteria, the AHL do not need an extracellular receptor. Instead, they diffuse into the cell. Upon reaching a critical concentration, the AHLs bind directly to LuxR-like proteins. This complex then activates the expression of specific genes.​1​

Pseudomonas aeruginosa

The bacterium Pseudomonas aeruginosa that we chose as our target pathogen is also capable of using quorum sensing. Among others, the QS-genes in P. aeruginosa are responsible for the virulence of the human pathogen.

Outbreaks of P. aeruginosa are often associated with water contamination and commonly cause nosocomial infections such as pneumonia.​3​ Furthermore, waterborne pathogens generally pose a high risk to human health, as 10% of all global illnesses are related to water contamination.​4​ Many of the P. aeruginosa isolates are antibiotic resistant. This is a major problem because the WHO classified P. aeruginosa as a critical antibiotic resistant bacterium for which new antibiotics must be developed.​5​

P. aeruginosa is capable of forming its own biofilms and even nesting in pre-existing biofilms like B. subtilis biofilms. For this reason, P. aeruginosa is sometimes difficult to detect in water because it hides in these biofilms where it can survive up to 28 days. In addition, P. aeruginosa can enter the so-called VBNC- viable but not cultivable – state. Here, the pathogen is no longer detectable by classical cultivation methods, but it is still capable of re-establishing its pathogenic activity.​3​

Quorum Sensing in Pseudomonas aeruginosa

Like other Gram-negative bacteria, Pseudomonas aeruginosa uses the LuxR/LuxI quorum sensing system with Lux-like proteins. Over 300 genes are controlled in a global expression system by quorum sensing. In this way, the production of virulence factors as well as biofilm formation are regulated. P. aeruginosa has two quorum sensing systems with different AHL, which are interconnected. First, the las system that is based on N-(3-oxododecanoyl)-homoserine lactone (3OC12-HSL), and second, the rhl system which is based on N-butanoyl-L-homoserine lactone (C4-HSL). In the las system normally the LuxI-like protein LasI synthesizes 3OC12-HSL. Other bacteria sense this AHL with the LuxR-like protein LasR. LasR functions as a transcription factor and forms a complex together with the AHL. Then, the complex binds specifically to the PLux-promoter to regulate gene expression.​6​ For our genetic circuit we chose to use the las system of P. aeruginosa with LasR.

In addition, P. aeruginosa uses the quorum sensing controlling repressor QscR to repress LasR at low AHL concentrations by forming a heterodimer. QscR is a LasR homolog with no cognate AHL synthetic enzyme. Instead, it also binds the AHL 3OC12-HSL. QscR can also control gene expression in P. aeruginosa directly by binding the promoter of PA1897.​7​ Besides LasR we want to use QscR as a second transcriptional factor that can be activated by the AHL 3OC12 -HSL.

3OC12-HSL is capable of binding both transcriptional factors, LasR and QscR. It is also the main and most studied quorum sensing molecule of P. aeruginosa and it is possible to detect P. aeruginosa successfully based on this QS molecule.​4,8​ This is why we decided to activate our B. subtilis sleeper cells as a Gram-positive bacterium with this signaling molecule. Since it has never been tested before whether 3OC12-HSL is compatible with B. subtilis cells we researched vastly and contacted several experts on this field. They gave us some positive insides and helped us develop our genetic circuit. Our research results are presented in the next section.

N-Acyl homoserine lactones and B. subtilis

With our sensing system we want to detect the presence of P. aeruginosa cells. Therefore, we focused on N-(3-oxododecanoyl)-homoserine lactone (3OC12-HSL) as a signaling molecule. 3OC12-HSL is an acyl homoserine lactone (AHL) (Figure 3) and the main quorum sensing molecule of P. aeruginosa.​9​ Most AHLs diffuse freely across its cell membrane and regulate gene expression by binding intracellularly to allosteric transcription factors (aTF).​9​ aTF change their conformation after binding such small molecules, which alters their affinity for operator DNA sequences. Wu et al. already successfully demonstrated the use of 3OC12-HSL to specifically detect P. aeruginosa by synthetic E. coli sensor cells.​4​ Since both bacteria are Gram-negative, they have comparable cell envelopes, which the quorum sensing molecules pass freely.

Figure 3. Structure of N-(3-oxododecanoyl)-homoserine lactone (3OC12-HSL), an acyl homoserine lactone (AHL).

We, however, want to use an AHL sensing system in B. subtilis, a Gram-positive bacterium with a cell membrane structure deviating from that of Gram-negatives. It has not been demonstrated that AHLs can diffuse freely across the membranes of B. subtilis, yet. But it has been reported that Gram-positive bacteria can interact with such AHL systems, for example bacteria of the genus Exiguobacterium, produce and release their own AHL.​10​ Furthermore, gene expression of Staphylococcus aureus or Listeria monocytogenes can be regulated by AHL.​11,12​ AHL are also known to have a cytotoxic effect on some Gram-positive bacteria, among them B. subtilis (EC50 = 31.1 µM, for 3OC12-HSL) which implies the ability to pass the cell membrane.​9​ However, toxic effects should not occur at the 3OC12-HSL concentrations our sensing system should react to.​4​

In one of our expert talks, Prof. Dr. Anke Becker assumed that AHL should be able to enter B. subtilis cells. She also explained to us that among longer-chain AHL, those with hydrophobic side chains enter more effectively into Gram-negative cells. This principle could be transferred to our implementation in Gram-positive B. subtilis. In doing so, she suggested a method to verify this by expressing a reporter gene under the control of an AHL induced promoter. We incorporated this suggestion into the design of our laboratory experiments.

Another possible challenge for our system could be that some Gram-positive bacteria possess lactonases.​13​ These degrade AHL molecules and function as quorum quenching systems.​14​ Such enzymes are not transported out of the cell but are active in the cytoplasm. This also indicates an intracellular presence of AHL.​13​ But, for our application, this could also prove as a disadvantage since our sensing system depends on the availability of 3OC12-HSL.

Some B. subtilis strains also encode AHL lactonases that degrade the quorum sensing molecule 3OC12-HSL. For example, the common AHL lactonase encoded by the gene aiiA.​15​ This lactonase hydrolyses different AHL molecules. Its KM value is 1.43 mM for 3OC10-HSL, which has a similar structure to 3OC12-HSL.​16​ The sensing system we want to use was reported to react to AHL concentrations in the nanomolar range when implemented in E. coli.​4​ This is three units smaller than the KM value of the AiiA lactonase for the hydrolysis of 3OC10-HSL. Therefore, an impact on our sensing system is not expected, premising the sensitivity of our sensing system in B. subtilis is similar to its sensitivity in E. coli.

Additionally, the gene ytnP can be found in the genome of the common laboratory strain B. subtilis 168. It shows sequence homologies to genes that encode quorum quenching lactonases.​17​ In an expert talk Prof. Dr. Fabian Commichau told us such quorum quenching lactonases are commonly produced under certain conditions (e.g., high salt concentrations). But since the expression product of ytnP is not further characterized, testing its effect on our system was included in the design of our laboratory experiments.

Thus, the results of our research indicate that the usage of AHL as signaling molecule for our sleeper cells should not turn out as problematic by undesired interactions with B. subtilis.

References

  1. 1. Miller MB, Bassler BL. Quorum Sensing in Bacteria. Annual Review of Microbiology. 2001 Oct:165–199. http://dx.doi.org/10.1146/annurev.micro.55.1.165. doi:10.1146/annurev.micro.55.1.165
  2. 2. Urry L, Cain M, Wasserman S, Minorsky P, Jackson R, Campbell N. Campbell Biology. 10th ed. Boston: Pearson; 2014.
  3. 3. Mena KD, Gerba CP. Risk Assessment of Pseudomonas aeruginosa in Water. In: Reviews of Environmental Contamination and Toxicology Vol 201. Springer US; 2009. p. 71–115. http://dx.doi.org/10.1007/978-1-4419-0032-6_3. doi:10.1007/978-1-4419-0032-6_3
  4. 4. Wu Y, Wang C-W, Wang D, Wei N. A Whole-Cell Biosensor for Point-of-Care Detection of Waterborne Bacterial Pathogens. ACS Synthetic Biology. 2021 Jan 26:333–344. http://dx.doi.org/10.1021/acssynbio.0c00491. doi:10.1021/acssynbio.0c00491
  5. 5. Tacconelli E, Magrini N. Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics. WHO. 2017 [accessed 2021 Oct 17]. https://www.who.int/medicines/publications/WHO-PPL-Short_Summary_25Feb-ET_NM_WHO.pdf
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  9. 9. Kaufmann GF, Sartorio R, Lee S-H, Rogers CJ, Meijler MM, Moss JA, Clapham B, Brogan AP, Dickerson TJ, Janda KD. Revisiting quorum sensing: Discovery of additional chemical and biological functions for 3-oxo-N-acylhomoserine lactones. Proceedings of the National Academy of Sciences. 2004 Dec 27:309–314. http://dx.doi.org/10.1073/pnas.0408639102. doi:10.1073/pnas.0408639102
  10. 10. Biswa P, Doble M. Production of acylated homoserine lactone by Gram-positive bacteria isolated from marine water. FEMS Microbiology Letters. 2013 Apr 2:34–41. http://dx.doi.org/10.1111/1574-6968.12123. doi:10.1111/1574-6968.12123
  11. 11. Qazi S, Middleton B, Muharram SH, Cockayne A, Hill P, O’Shea P, Chhabra SR, Cámara M, Williams P. N-Acylhomoserine Lactones Antagonize Virulence Gene Expression and Quorum Sensing in Staphylococcus aureus. Infection and Immunity. 2006 Feb:910–919. http://dx.doi.org/10.1128/iai.74.2.910-919.2006. doi:10.1128/iai.74.2.910-919.2006
  12. 12. Naik MM, Bhangui P, Bhat C. The first report on Listeria monocytogenes producing siderophores and responds positively to N-acyl homoserine lactone (AHL) molecules by enhanced biofilm formation. Archives of Microbiology. 2017 Jul 31:1409–1415. http://dx.doi.org/10.1007/s00203-017-1416-8. doi:10.1007/s00203-017-1416-8
  13. 13. Pan J, Huang T, Yao F, Huang Z, Powell CA, Qiu S, Guan X. Expression and characterization of aiiA gene from Bacillus subtilis BS-1. Microbiological Research. 2008 Nov:711–716. http://dx.doi.org/10.1016/j.micres.2007.12.002. doi:10.1016/j.micres.2007.12.002
  14. 14. Grandclément C, Tannières M, Moréra S, Dessaux Y, Faure D. Quorum quenching: role in nature and applied developments Camara M, editor. FEMS Microbiology Reviews. 2015 Oct 1:86–116. http://dx.doi.org/10.1093/femsre/fuv038. doi:10.1093/femsre/fuv038
  15. 15. Molina L, Constantinescu F, Michel L, Reimmann C, Duffy B, Défago G. Degradation of pathogen quorum-sensing molecules by soil bacteria: a preventive and curative biological control mechanism. FEMS Microbiology Ecology. 2003 Jul:71–81. http://dx.doi.org/10.1016/s0168-6496(03)00125-9. doi:10.1016/s0168-6496(03)00125-9
  16. 16. Wang L-H, Weng L-X, Dong Y-H, Zhang L-H. Specificity and Enzyme Kinetics of the Quorum-quenching N-Acyl Homoserine Lactone Lactonase (AHL-lactonase). Journal of Biological Chemistry. 2004 Apr:13645–13651. http://dx.doi.org/10.1074/jbc.m311194200. doi:10.1074/jbc.m311194200
  17. 17. Schneider J, Yepes A, Garcia-Betancur JC, Westedt I, Mielich B, López D. Streptomycin-Induced Expression in Bacillus subtilis of YtnP, a Lactonase-Homologous Protein That Inhibits Development and Streptomycin Production in Streptomyces griseus. Applied and Environmental Microbiology. 2011 Nov 18:599–603. http://dx.doi.org/10.1128/aem.06992-11. doi:10.1128/aem.06992-11
igem@bio.tu-darmstadt.de

We are the 2021 iGEM Team of the TU Darmstadt. This year we focused on creating a self-protecting biofilm based on Bacillus subtilis "sleeper cells".

Eppendorf hilgenberg Zymo Research New England Biolabs Inc.
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