Team:UNICAMP-EMSE Brazil/Project

Abstract

The human immune system comprises a wide range of cells and mechanisms finely regulated to maintain body's homeostasis against eventual threads. However, pathological conditions, such as stress and autoimmune diseases, can cause imbalances in its action rendering higher susceptibility to potential pathogens. This imbalance is ultimately observed in the biased differentiation of naive T-CD4⁺ Lymphocytes towards T-lymphocyte "helper" class 1 (T_h1), in autoimmune diseases, or 2 (T_h2), in stressful condition, favoring cellular or humoral adaptive responses, respectively. This pathological imbalance renders the organism more susceptible to the onset of diseases caused by microorganisms. Thus, a mechanism that counteracts this imbalance is highly desirable. The aim of our project is to use the ability of some microorganisms to sense changes in the host´s body homeostasis, used by them as a signal to trigger expression of virulence factors, to counteract this pathological imbalance. In this sense, the ability of some gut-inhabiting bacteria to sense cathecolamines, which can reach high levels on the gastro-intestinal tract during stress, will be used to trigger the production of IL-12, a potent activator of T_h1, to counteract the preferential differentiation towards T_h2. On the other hand, the ability to sense Nitric-Oxide (NO), a compound released at higher levels in inflammatory conditions, will be used to trigger the production of IL-10, a cytokine with a potent anti-inflammatory action. To switch between both responses, a mechanism that can break down the opposite signaling molecule on the recognition of the first signal will be developed. The microorganism bearing these devices will thus be able to restore the appropriate balance between T_h1 and T_h2 cells in the host's organism, thus counteracting harmful outcomes of this condition and, hence, improving human life quality.

Project Description

Stress can be understood as the perception of a potentially dangerous state for homeostasis maintenance, triggering a series of physiological responses. Any stressor or threat to stability of the organism is counteracted by adaptive responses. The effectors that trigger the physiological responses to stressful conditions are the corticotropin-release hormone (CRH) and the locus ceruleus-noradrenaline (LC-NA)/sympathetic neurons of the hypothalamus. The activity of these neurons regulate the activation of HPA (Hypothalamic-Pituitary-Adrenal) axis and the Sympathetic Nervous System that result in systemic elevations in levels of glucocorticoids (GCs) and cathecolamines (CAs), like epinephrine and norepinephrine.

There has been convincing evidence that the GCs and CAs at the high levels achieved in stress can influence the immune response in an important way, acting as an immune suppressor agent of cellular immunity (Elenkov et al., 1999). Specially, CAs shift differentiation of naive T CD4+ Lymphocytes into the Th2 (T-lymphocyte "helper" class 2) lineage, via inhibition of the production of the cytokine IL-12 (Interleukin 12, a potent activator of differentiation into Th1 Lymphocytes lineage) and induction of the production of the cytokine IL-10, as was shown in a study using human blood cultures stimulated with lipopolysaccharide (LPS) ex vivo (Elenkov et al., 1996). This effect can be completely prevented using propanolol, an antagonist of β-Adrenoceptors. Together with GCs, CAs lead to a suppression of antigen-presenting cells (APCs) and Th1 cells. The latter mediates the cellular immune response. This polarization affects the immune response, causing imbalance to an increased susceptibility to disease and immunosuppression.

The main purpose of Jedi bacteria is the victory in the Stress Wars, through producing IL-12 when the host is under stress to avoid immunesupression caused by Catecholamines and to avoid mad bacteria to attack the host in this susceptibility moment

Recently, it was demonstrated that several bacterial pathogens (such as Escherichia, Yersinia and Pseudomonas) can sense host cues and so activate expression of their virulence genes. In the case of host stress, bacteria can sense the signal that defines a susceptible condition of the host immune system (characterized by high levels of CAs and GCs). In other words, such pathogens can sense and respond to increasing levels of CAs as a way to recognize the host environment and to promote the expression of virulence and growth factors, via a Bacterial Adrenergic Receptor protein, QseC, and its signaling pathway (Reading et al., 2009). The control of this bacterial system is supposed to be a new way to prevent diseases.

The use of probiotics has been explored extensively so far, however, there is an increasing pressure for improvements in the formulation of its composition. The use of genetic engineering and rational design of genetically modified organisms (GMOs) has been considered as an alternative against onset of stress and diseases. For example, Steidler et al. (2000) used a genetically modified strain of Lactococcus lactis expressing the gene for interleukin IL-10 to treat mice presenting irritable bowel syndrome (IBD), by means of bacteria being injected directly into the gastric lumen of mice.

Our project aims to produce a probiotic capable of restoring the balance between Th1 and Th2 immune response, with the JEDI BACTERIA, in order to improve the quality of life and reduce the effects triggered by stress, such as induction of virulence genes of Bacterial Pathogens. Through expression and secretion of IL12 and degradation of NO under high levels of Cathecolamines, we hope we would be able to induce Th1 lineage of Lymphocytes and suppress Th2, declaring a great 'War' against the Stress.

Complete description of devices

Device 1: Adrenaline sensor/IL-12 producer

Figure 1: Representation of Jedi bacteria containing device 1 (able to sense high levels of CAs and respond by producing IL-12) and device 3 (secretion system device, represented as the orange cylinder).

Figure 2: Another representation of device 1 (able to sense high levels of CAs and respond by producing IL-12) and device 3 (secretion system device).

Device 2: NO sensor/IL-10 producer

This device is a modification of Stanford team anti-inflammatory device (iGEM 2009 - https://2009.igem.org/Team:Stanford/ProjectPage), which comprises SoxR gene (BBa_K223047) under control of a Constitutive Promoter (BBa_J23119) and SoxS promoter (BBa_K223001 – deleted part), coupled to human IL-10 gene (sequence designed by the team, improved for bacterial expression).

It acts by recognizing an inflammatory signal (NO - nitric oxide) coupled to IL-10 transcription activation. The team chooses IL-10 to impair inflammatory response mediated by Th1 overstimulation (due to Device 1 action), to reach an equilibrium between immune stimulation (Th1) and suppression (Th2).

Nitric oxide is generated by immune cells (monocytes, macrophages and neutrophils) as a part of immune response, since it as signaling molecule and is toxic to bacterial pathogens. These cells possess an inducible nitric oxide synthase (iNOS), which is activated by cytokines as tumor necrosis factor (TNF) and interferon-gamma (IFN-γ) released by macrophages and regulatory T-cells (Hibbs et al 1988; Wink et al 1991).

IL-10 is an anti-inflammatory interleukin, produced mainly by monocytes. It is responsible for down-regulating the synthesis of pro-inflammatory cytokines like IFN-γ, IL-2, IL-3, TNFα (Th1 cytokines), class II MHC, and co-stimulatory molecules on macrophages. IL-10 also displays potent abilities to suppress the antigen presentation capacity of APCs (Antigen Presenting Cells), and it is also responsible for enhancing B cell survival, proliferation and antibody secretion. In this scenario IL-10 favors Th2 response (Mocellin et al 2004, Elenkov & Chrousos, 1999).

Some studies suggested that IL-10 acts as immunoregulator in intestinal tract. For example Steidler et al (2000) used a genetically modified Lactococcus lacti expressing IL-10 to treat mice with Inflammatory Bowel Disease (IBD), and the inflammatory response signals were reduced in 50%. IBD is an autoimmune disease which, unlike the immune response found in intestinal tract due to stress, involves a Th1 polarization and inflammation.

As NO is toxic to bacteria, many bacterial pathogens have evolved mechanisms for nitric oxide resistance. For example, E. coli can naturally respond to redox signals through a superoxide stress system composed by SoxR gene and SoxS promoter. The [http://partsregistry.org/Part:BBa_K554003 SoxR] gene encodes the SoxR transcription factor, which is activated by NO. Thus, in the presence of NO, SoxR activates transcription of the gene regulated by [http://partsregistry.org/Part:BBa_K554000 SoxS promoter] (Hidalgo et al, 1998).

Figure 3: Representation of Jedi bacteria containing device 2 (able to sense high levels of NO and respond by producing IL-10) and device 3 (secretion system device).

Device 3: Secretion system

So our bacteria have sensed the stress, and have reacted to this by successfully producing interleukins. But there’s no use in storing our interleukins, since they can only act outside the cell, directly on our immune system. Therefore we had to add to our Stress Warrior a system that allows its secretion.

We chose to use the ABC protein-mediated transporter, typical of Gram-negative bacteria, just like our E. coli. It’s composed of 4 parts: the C-terminal signal sequence of alpha-hemolysin (HlyA), the two specific translocator proteins HlyB and HlyD and the outer membrane protein TolC.

HlyB acts as a ATP-binding cassette, and recognizes the substrate via its secretion signal (like HlyA) and is responsible for the specificity of the secretion process. HlyD acts as a membrane fusion or adaptor protein, consisting of a short cytoplasmic domain at the N-terminus followed by a membrane anchor and a large periplasmic domain; it is believed to establish specific links between the outer and the inner membrane components of the system. Finally, TolC is a specific outer membrane protein, which forms a long channel throughout the outer membrane and the periplasm, largely open towards the extracellular medium. These 3 components will be kept expressed in our bacteria at all times.

HlyA, the signal sequence, seems to interact with the cytoplasmic region of the pre-formed HlyB–D complex. After the binding of the HlyA secretion signal by the HlyB–D complex, HlyD induces the interaction with TolC. To be effective, only the last 50-60 aminoacids of the C-terminal of Hly-A are required, so we can use it as a fusion protein that is attached to the interleukin and allows it to be secreted. Besides, HlyA is itself a weak antigen, turning it harmeless to the immune responses we are trying to interfere with. References: Tzschaschel (1996), Delepelaire (2004) and Gentschev (2002).

You can see a representation of this device acting in the schema below:

Figure 4: Representation of device 3, the protein secretion system, in a Jedi bacteria that contains Device 1 (Adrenaline sensor/IL-12 producer). To export a protein, the bacteria must have the HlyD, HlyB and TolC proteins and the target protein must have a signal sequence (HlyA tail). In this case, the target protein to be secreted is IL-12.

The Experiments

Check our device testing experiments at Results page and the day-to-day work done to built the devices at our Notebook page.

Applications

• It’s really well known how stressful is our life and how much we are affected by this. But what is Stress exactly? Stress is a word with a broad meaning, but generally it relates to a set of symptoms and reactions showed by our bodies in response to external factors that bring us pressure, anxiety or any emotional imbalance.

• It is not exactly something easy to control!

• Can you simply stop caring about being fired? About your tests next week? Or can you decide to ignore the iGEM deadlines? Well, we just need to accept that stress is part of our lives and learn how to deal with it.

• Regarding this, our team wanted to develop a way to reduce stress symptoms that directly affect our health, and our focus is the immune system. The major application we expect to get from Stress Wars would be the launching of a stress-beating-probiotic.

• In our view, stress is present in our every day life, so a probiotic, which is consumed on a regular basis, could keep the immune balance. Responding to both adrenaline and NO stress, it would cover the two major sources of imbalance, by over expressing interleukins and redirecting the immune response without adding exogenous substances or medication. This could guarantee a long-term immune balance that can only favor health.

• Besides, the idea of affecting the immune balance is really useful and has various potential applications, including the optimization of the response to various vaccines and the treatment of several diseases that are known to be caused by immune imbalances, such as cancer, HIV, type I Diabetes and Multiple Sclerosis.

REFERENCES

Elenkov IJ, Chrousos GP. Stress Hormones, Th1/Th2 patterns, Pro/Anti-inflammatory Cytokines and Susceptibility to Disease. Trends Endocrinol Metab. 1999 Nov;10(9):359-368. [http://www.ncbi.nlm.nih.gov/pubmed/10511695 Link to Pubmed ]

Elenkov, I.J., Papanicolaou, D.A., Wilder, R.L. and Chrousos, G.P. (1996) Modulatory effects of glucocorticoids and catecholamines on human interleukin-12 and interleukin-10 production: clinical implications. Proc. Assoc. Amer. Physic. 108, 374–381. [http://www.ncbi.nlm.nih.gov/pubmed/8902882 Link to Pubmed ]

Reading NC, Rasko DA, Torres AG, Sperandio V. The two-component system QseEF and the membrane protein QseG link adrenergic and stress sensing to bacterial pathogenesis. Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5889-94. Epub 2009 Mar 16. [http://www.ncbi.nlm.nih.gov/pubmed/19289831 Link to Pubmed]

Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, Fiers W, Remaut E. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10, Science 289. 2000. 1352–1355. [http://www.ncbi.nlm.nih.gov/pubmed/10958782 Link to Pubmed]

Barbara D. Tzschaschel, Carlos A. Guzmán,, Kenneth N. Timmis and Victor de Lorenzo. An Escherichia coli hemolysin transport system-based vector for the export of polypeptides: Export of shiga-like toxin IIeB subunit by Salmonella typhimurium aroA. Nature Biotechnology 14, 765 - 769 (1996) [http://www.nature.com/nbt/journal/v14/n6/abs/nbt0696-765.html Article link]

P. Delepelaire. Type I secretion in Gram-negative bacteria. Biochimia et Biophysica Actca 1694, 149-161 (2004) [http://www.ncbi.nlm.nih.gov/pubmed/15546664 Link to PubMed]

Ivaylo Gentschev, Guido Dietrich and Werner Goebel. The E. coli α-hemolysin secretion system and its use in vaccine development. Trends in Microbiology 10, 39-45 (2002) [www.ncbi.nlm.nih.gov/pubmed/11755084 Link to PubMed]