Aashi Agarwal, Samuel J. Dillon, Michael Hu, Xiaoye Jin, Apurva Joshi, Rohit Kommuru, Song Leav, Anya Mittal, Hannah Tandang, Henry Tshabalala, Grace Xu, Amanda Zhang
BioBuilderClub, Acton-Boxborough Regional High School, Acton, Massachusetts, USA
Reviewed on 8 May 2021; Accepted on 28 June 2021; Published on 25 October 2021
With help from the 2021 BioTreks Production Team.
Keywords: Legionella detection, bioluminescence, quorum sensing, clean water
Authors are listed in alphabetical order. Aaron Mathieu mentored the group. Please direct all correspondence to amathieu@abschools.org.
Background
Legionnaires’ disease is a severe type of pneumonia that occurs when water droplets containing Legionella are inhaled and make their way into lungs (Centers for Disease Control and Prevention, 2021a). Normally, macrophages—a type of white blood cell—would be able to attack and break down invading bacteria and viruses; however, Legionella spp. avoids this by attacking the macrophages themselves (Centers for Disease Control and Prevention, 2021b). Inside these cells, Legionella survives and replicates, eventually taking over the macrophage and destroying it (Segal et al., 1998). This can cause tissue damage and inflammation in the lungs, which blocks the passage of oxygen, causing many negative effects. Initially, symptoms may include cough, fever, muscle aches, and headaches. People with the disease experience varying long-term effects as well. Sixty-six percent suffered from neurologic symptoms like memory loss and seventy-five percent from fatigue (Dooner, 2021). If not treated properly, the disease can cause multi-organ failure, eventually leading to death (Dooner, 2021). Because Legionnaires’ is a lung disease, people who smoke, have underlying lung illnesses, or are over fifty years of age are more at risk (Dooner, 2021).
There are more than 58 species of Legionella, with 25 pathogenic species, the most common pathogenic species being Legionella pneumophila. However, some other Legionella spp. can cause Pontiac fever, a milder infection. This is not a type of pneumonia, so while it has most of the same symptoms as Legionella, it lacks those which are pneumonia related. Its symptoms are less severe, typically lasting less than a week (State of Hawaii, Department of Health, 2019).
Legionnaires’ disease is often perceived as rare, but this is not the case. The false perception is caused by the rarity with which cases and even smaller outbreaks make the news (HC Info, 2021). According to the Center for Disease Control and Prevention (CDC), each year, between 8,000 to18,000 people are hospitalized with Legionnaires’ disease in the United States (“Legionnaires Disease”, n.d.). Because approximately 5-10% of people who contract the disease die, between 400 and 1800 deaths occur in the US each year. The CDC also states that “many infections are not diagnosed or reported, so the number affected may be higher” (“Legionnaires Disease”, n.d). It is often mistaken for another type of pneumonia, because recognizing it as Legionnaires’ takes special testing (Centers for Disease Control and Prevention, n.d.).
Legionella are also Gram-negative bacteria, meaning they are less susceptible to antibiotics than Gram-positive bacteria because Gram-negative bacteria have an outer lipid membrane, making them more dangerous. Taken together, there is a need for continued, if not increased, testing for Legionella.
When they arrive at a host’s alveolar macrophage via their polar flagellum, they inject effector molecules through the Dot/Icm T4SS system (Winn, 1996). This bacterial secretion system allows Legionella to deliver DNA or proteins into eukaryotic cells to carry out replication and certain activities to evade the immune response (Copenhaver et al., 2014). These effector molecules allow for the creation of a Legionella-containing vacuole that provides the ideal growing environment and protects Legionella against being destroyed. The Legionella bacteria then go on to multiply and spread within the host (Ziltener et al., 2016).
The table below summarizes the most common current solutions, their strengths, and their weaknesses.
Table 1. The three widely used tests for Legionella.
Our goal was to engineer a system that can improve on these weaknesses: a system that is accurate, fast, and easy to use. Now that the design is completed, we hope that the use of Legionella’s quorum sensing system will provide high accuracy, reasonable speed, and that—through experimentation with the application of our system—implementing the system would be highly simplistic. Further descriptions of our system’s advantages are in the discussion section.
Systems Level
This system is designed to detect the presence of Legionella spp. in water using a genetically engineered chassis. The system will be added to water samples with an experimentally determined amount of our modified chassis. Additionally, the system detects Legionella’s specific quorum sensing molecule (LAI-1) with Legionella’s own quorum sensing system (Lqs), which is included in our chassis (Personnic et al., 2018).
Quorum sensing (QS) is a bacterial system in which specific signal molecules are detected by corresponding receptor proteins at both intraspecies and interspecies levels. QS allows bacteria to determine population densities and alter gene expression accordingly (Rutherford & Bassler, 2012). Our QS pathway activates a bioluminescent reporter that produces light and induces the production of more LAI-1 to amplify the response.
Device Level
We chose to use Escherichia coli HB101 as our chassis (Figure 1). It is a freshwater bacterium and laboratory strain that can be genetically engineered to express detection and reporting systems (Podstawka, 2020). Legionella also lives in freshwater, so having a freshwater bacterium as our chassis is ideal because it eliminates the possibility of our chassis drowning when in the same environment with Legionella. E. coli HB101 also does not produce bioluminescence naturally, which will reduce noise from natural bioluminescence during experimentation. A plasmid containing the Lqs system genes, which produce proteins involved in Legionella’s quorum sensing system (Lqs), and the luxCDABEG operon, which produces luciferase and the luciferin that react to create bioluminescence (Figure 3), will be inserted into E. coli HB101.
Parts Level
Our E. coli HB101 is engineered with parts from Lqs to detect Legionella. Legionella produces a α-hydroxy ketone signaling molecule called LAI-1 (Legionella autoinducer-1, or 3-hydroxy pentadecane-4-one) through the autoinducer synthase LqsA (Hochstrasser & Hilbi, 2017). On the membrane of the engineered E. coli, the sensor kinase LqsS then detects LAI-1 (Figure 2).
The detection of Legionella using the LqsS sensor kinase will stimulate LqsR in the engineered E. coli HB101 through phosphorylation. LqsR then inhibits an lqsA gene repressor, resulting in the transcription of lqsA (Figure 2). lqsA produces LqsA proteins, which synthesize additional LAI-1 molecules that amplify the original signal (Hochstrasser & Hilbi, 2017).
Additionally, LqsR will be further designed to activate the transcription of luxL, to produce LuxL proteins, which synthesize an acylated homoserine lactone (AHL), specifically 3-oxo-C6-HSL molecules (Figure 3). These AHL molecules bind to the LuxR protein, which is produced by the luxR gene, to form a complex (Dunlap, 2009). The AHL and LuxR protein complex then binds to the luxCDABEG promoter to activate the transcription of the lux operon by RNA polymerase. The luxA gene and luxB gene lead to the production of the protein luciferase. Additionally, the luxC, luxD, and luxE genes code for Fatty Acid Reductase, and the luxG gene codes for Flavin Oxido-Reductase (Figure 3). The luciferase protein catalyzes the oxidation of the luciferin FMNH2, which is produced by Flavin Oxido-Reductase’s reduction of FMN, and the long chain aldehyde RCOH, which is produced by Fatty Acid Reductase’s reduction of RCOOH, to create bioluminescence (Brodl et al., 2018; Miyashiro & Ruby, 2012).
Safety
The most prevalent safety concern with the creation and testing of our system is the use of Legionella pneumophila. As stated in the “Background” section, exposure to this bacterium can cause Legionnaires’ disease, which is an often deadly type of pneumonia. Most of its survivors are left with long-term symptoms as a result (Legionella.org, 2016). In the worst cases, the death rate can be as high as 80% for immunosuppressed people who are not treated, and overall it kills 5-10% of people infected (World Health Organization, 2018). We would follow lab safety procedures according to Biosafety Level 2 (BSL-2) guidelines to reduce the risk of infection.
We would start by testing the system with LAI-1 and not Legionella itself (outlined in the Next Steps section), which removes the need for the required BSl-2 lab until we want to test our system with actual Legionella. We would then need to partner with a university or industry lab, as these standards cannot be met by a high school level laboratory.
Our chassis E. coli HB101 is a laboratory strain, and thus would only need a BSL-1 lab rating to use. It is not as dangerous as using Legionella, but we would still have to follow lab safety precautions. Since both of these bacteria cannot be released into the environment, they should only be used in a lab setting or at water treatment plants to test for L. pneumophila with the proper precautions. To reduce risk to surrounding populations and users of our device, we will test multiple applications of our system to find the safest and most reliable one.
Discussions
This system is designed to quickly and easily detect Legionella spp. in locations commonly associated with its presence, like water towers, which will allow for better control and prevention of future outbreaks of the deadly Legionnaires’ disease. This device would be a better solution in many cases compared to the current detection methods, as it may be quickly tested on contaminated water, without requiring a great amount of prior experience. As shown in Table 1, the current solutions for detecting Legionella each have one or more of the problems this device aims to solve, which will be determined through experimentation. For example, a dirty water sample messes up the PCR method (Table 1), while our engineered E. coli would not be affected by a dirty sample. This means less preparation of the sample would be needed before testing, which saves time and reduces necessary expertise.
Finally, using the quorum sensing method will allow for our device to detect all types of Legionella because all Legionella spp. use the Lqs system. This is an advantage over some current detection methods, because not all of them can detect all species of Legionella. We expect that our system would also be relatively accurate because this quorum sensing system is specific to Legionella, meaning bioluminescence would not occur if some other bacteria’s QS molecule were to come near our chassis.
A possible issue with our implementation in using samples to detect Legionella is detecting low concentrations of Legionella in water. Since the amount of Legionella in water towers varies, a small sample being tested from a large water tower may provide an inaccurate representation of the whole tower. Future research may be done to figure out the minimum amount of sample needed to provide accurate Legionella detection.
To sense LAI-1, we plan to use the LqsS membrane-bound sensor kinase engineered in E. coli HB101 (Figure 1). For our sensing to work correctly, Legionella’s signaling molecule must be able to pass through the cell membrane of the E. coli HB101 cells. LAI-1 is mostly hydrophobic and small, and thus should be able to easily diffuse through the membrane (Personnic et al., 2018). This device contains genetic modular elements from the specific pathogen to be detected (the parts of the Lqs system in Figure 2). We aim to allow the detection of multiple common/deadly pathogens using the same device, but with different modules. This will allow the device to be used for a longer period of time at facilities, and as new pathogens arise. Additionally, this will allow costs to be reduced as facilities do not need to replace the whole device if new pathogens are to be tested, or if a part of the device malfunctions. Future improvements will focus on increasing accuracy and speed of detection.
Additionally, new deadly pathogens that require a quick test will automatically present an improvement opportunity. Future improvements to this device are linked to the modularity of the device; new improved modules may be added as an extra module, which will make future improvements easier to add. Further discussion of future improvements is in the “Next Steps” section.
Next Steps
Having done most of the background research, the next step would be to build the system and test it in a lab setting. As detailed in the “Safety” section, working with Legionella requires a BSL-2 lab rating. Thus, we would start by testing our system with only the quorum sensing product of the Legionella to bypass the need for a lab with a BSL-2 rating until we want to experiment with actual Legionella.
We plan on testing the detection and bioluminescence pathways (Figure 2 & Figure 3 respectively) separately so that it would be easier to determine which pathway does not work as expected. We would start by adding our chassis—engineered to only express the necessary parts up until the production of AHL and LAI-1 (Figure 2)—into a sample of water. This method may need the additional production of green fluorescent protein (GFP) to observe, but this would be a simple addition to the system since making LqsR also activates a GFP pathway. We would first test the detection pathway by itself for the production of AHL and LAI-1. In this way, we will detect false negative outcomes (i.e. no production of AHL and LAI-1 in a sample that has Legionella’s QS molecule LAI-1) are due to problems with the detection pathway and not the bioluminescence pathway. Generally, we would test this by adding our engineered E. coli into positive control samples (those which we know have LAI-1), and once it works on those samples, we would test the system on unknown water samples that potentially contain real Legionella.
Once the detection system can accurately produce AHL in both scenarios, we will combine the bioluminescence and detection pathways to test if our bioluminesce pathway works. We expect to be able to see the bioluminescent output with our bare eyes, however we may find that the strength of the signal may need to be increased. We could do this by increasing the output of our amplification system by making each E. coli produce a larger number of new LAI-1 molecules after it detects Legionella.
After testing that the system works, we plan to experiment on improving the sensitivity of the detection and the time detection takes. There are non-pathogenic strains of Legionella that may cause our system to detect false positives. Thus, in future, we will determine a method of differentiating the false positives from samples which actually contain pathogenic Legionella. Additionally, we would experiment with making the strength of the bioluminescent light proportional to the concentration of Legionella: the higher the concentration of Legionella, the brighter the light should be. To determine the time our detection system takes, we would first test our system with only LAI-1 molecules and then with Legionella. Once we have established a baseline with our original system, we would tweak the system, possibly by changing the parts in the pathway or the environment of our application, to optimize the time detection takes.
When creating the application of our system, we also need to determine how many E. coli HB101 would need to be present in the system, and the relationship between the number of E. coli HB101 present and light intensity produced by the accumulation of luciferase. We are also considering a cell-free system, which would not dilute our samples as much as adding many E. coli cells, giving us a higher concentration of Legionella, which could improve our accuracy in testing. Once the Legionella is detected, we would then need to eliminate it. The simplest way to do this would be to boil the water and kill the Legionella, but we plan on exploring methods of bacterial elimination as well.
Besides detecting Legionella, there are other quorum sensing bacteria that we could easily detect by switching out the membrane-bound sensors and response regulators to match the quorum sensing molecule of the bacteria we are targeting. Vibrio cholerae is a great example because it has a similar quorum sensing system to Legionella, with similar parts that could be switched out.
Author Contributions
All authors contributed to the “Abstract.” X.J., A.M., and H.Ts. wrote the “Background” section. A.A., S.D., A.J., and A.Z. researched the detection system, and S.D. wrote the “Systems Level,” “Device Level,” and “Parts Level” for the detection system. A.A., X.J., R.K., S.L., H.Ta., and A.Z. researched the bioluminescence pathway, and A.A. wrote the “Device Level,” and “Parts Level” for the bioluminescence pathway. S.L. wrote the “Safety” section. M.H. wrote the “Discussions” section. H.Ta. wrote the “Acknowledgements” section. A.J., H.Ta. wrote the “Next Steps” section. S.D. made the “References” section.
Acknowledgements
We would like to thank our teacher Mr. Mathieu and our mentor William Beeson for providing us with expert advice and giving us their immense support throughout the year.
This project was accomplished through participation in the BioBuilderClub, an after-school program organized by the BioBuilder Educational Foundation. The BioBuilderClub engages high school teams around the world to combine engineering approaches and scientific knowledge to design, build, and test their project ideas using synthetic biology.
References
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