Team:UTokyo/Description

YEAST-AID

Description

Index
  1. 1. Project Background
    1. 1.1. Increasing Demand for Nursing Care in an Aging Population
    2. 1.2. Problem of Bedsores and Their Care
  2. 2. Project Goals
  3. 3. Design
    1. 3.1. Overview
    2. 3.2. Oxygen sensing
    3. 3.3. Defensin secretion
    4. 3.4. Pathogen detection

What can we do to extend a healthy life expectancy? —— In our steadily aging society, the establishment of innovative tools to improve medical and nursing care is one answer to this question. This year, we are developing a multifunctional adhesive bandage using synthetic yeasts which will open up a possibility of home healthcare.

Project Background

Increasing Demand for Nursing Care in an Aging Population

The aging of Japan’s population is progressing at the most rapid pace in the world, and it is estimated that the rate of elderly people over the age of 65 will reach nearly 40% by 2050 [1]. Not only Japan but also many other countries all over the world are going to face the same problem in the near future. The number of elderly people is projected to double to 1.5 billion globally in the next 30 years [2].

Figure showing the increasing number of Japan’s elderly people

Fig. 1 The observed and estimated number of elderly people in Japan

The increasing number of elderly people requires more and more support in the medical and nursing sectors. One of the strongest demands is home care, with many people choosing to receive medical treatment in a familiar environment, either by choice or out of necessity. However, these needs are not adequately met because of a serious shortage of caregivers and healthcare professionals. What is worse, the spread of COVID-19 has aggravated this situation: Many people decided to receive care at home to avoid the risk of infection. Healthcare professionals may not be able to visit care-receivers frequently due to a lockdown of the city. The scarce access to caregivers leads to a disastrous problem when a care-receiver has a medical condition that is progressive and requires regular care by professionals.

Problem of Bedsores and Their Care

Our project focuses on bedsores, also known as pressure ulcers, which are one of such medical conditions. Bedsores are serious injuries to the skin and underlying tissue caused by prolonged pressure. They often happen to elderly people who spend a lot of time in bed and diabetic patients who take a long time in wound recovery. Without correct treatment, the damage can progress to deeper tissue under the skin and become inflamed by infection from bacteria. About 1% of those who receive care at home were reported to have bedsores in Japan [3]. In the case of other countries, the percentage amounts to nearly 11% in the US [4]. In 2015, nearly 1.2 million cases of bedsores were reported to occur in the US hospital [5]. Patients with bedsores require more than twice as much time and money in hospitals as those who do not have them.

Figures comparing time and money bedsore patients and non-patients spend

Fig. 2 Hospitalization periods (left) and costs (right) of patients with or without bedsores

Currently, the care of bedsores is mainly conducted using wound dressings [6]. These applied bandages keep the wound in a moist environment and help it heal. However, this method puts a burden on caregivers because frequent replacement of bandages is needed. The process of putting on new dressings takes about one hour and mostly requires the help of caregivers. Moreover, it is difficult to monitor the condition of the wounds underneath without medical professionals. Bedsore treatment is especially difficult in that it requires different types of medicine according to the condition of the wounds, and inappropriate use of dressing material can be a hotbed of bacterial infection.

Through the course of our integrated human practices, we realized the seriousness caring for bedsores. Therefore we decided to use the power of synthetic biology to create a device that is able to manage the wounds easily and effectively.

Project Goals

The goal of YEAST-AID is to develop a multifunctional wound dressing using synthetic yeasts. The bandage applied on the bedsore makes it possible to correctly monitor the condition of wounds and sanitize the skin. More specifically, our smart bandage changes colors when the wound has healed or bacterial infection has happened. Yeasts inside of the bandage also secret antimicrobial peptides to prevent the cause of inflammation. These functions allow bedsore patients to effectively receive care from non-professionals, such as their family members. In essence, YEAST-AID is a technology-driven solution to improve the care of bedsores in an aging population.

Design

Overview

Why did we choose yeast?

While Escherichia coli is generally easier to handle, we chose yeast Saccharomyces cerevisiae as the chassis for our project, for the following three reasons:

  1. S. cerevisiae has a mechanism for extracellular secretion.
  2. S. cerevisiae can be easily recovered from a dry dormant state with water, which is useful in implementation.
  3. S. cerevisiae is familiar as a food ingredient like bread and alcohol, so there are few psychological barriers for users.
    The ease of handling and familiarity with yeast make social implementation of our product simple and easy.

Function

The wound dressing we are developing has the following three functions, which will enable non-professionals to provide easy and effective pressure ulcer care.

Wound monitoring by oxygen sensing

The goal of this function is to monitor wound healing by sensing changes in the oxygen concentration in the wound.
Generally, it is not easy to quantitatively monitor the state of a wound. In our project, we propose an implementation that focuses on oxygen concentration.
According to previous research, the oxygen concentration in a wound changes depending on its healing state [17][18]. This is because oxygen consumption increases in the wound area, resulting in a normal oxygen state in a healed wound and a hypoxic state in a non-healed wound. By detecting this change in oxygen concentration, the healing state of the wound can be monitored.

Defensin secretion to keep wound clean

Bacterial infections in bedsores can be minimized with the use of the antimicrobial peptide called “Defensins”.
One of the major problems that arises in the management of bedsores is infection by bacteria, particularly Pseudomonas aeruginosa and Staphylococcus aureus . By secreting Defensins from the yeast, we aim to prevent the infection.

Pathogen detection to prevent a wound infection

The goal of this function is to detect bacterial infections in bedsores as soon as possible and alert the patient to the need for special treatment.
Bacterial infections, caused especially by P. aeruginosa and S. aureus, are among the most common problems in bedsore treatment. Particularly, biofilm formation in wounds caused by P. aeruginosa is a major problem that requires immediate treatment. Therefore, in our project, we build a system to detect P. aeruginosa infection of wounds by monitoring the intercellular communication signals.

Fiber-like hardware

Yeast, which has the three functions described above, needs to be implemented in an appropriate form so that it does not leak into the wound while accurately monitoring and treating the wound. This is made possible by using “cell-fiber,” a fiber-like cell encapsulation material [21]. The genetically modified yeast is encapsulated in this fiber-like gel.
For more details, please refer to the Hardware page.

Oxygen sensing

We have developed a gene circuit for sensing changes in oxygen concentration using the aerobic-anaerobic switching mechanism of S. cerevisiae.
S. cerevisiae has a pathway for switching between aerobic and anaerobic respiration, called the Pasteur effect, and many factors are involved in the regulation [15]. Among the factors involved in this complex pathway, we focused on a group of genes that are regulated by ROX1.
ROX1 is a gene whose expression is activated under aerobic conditions and functions as a repressor. Under aerobic conditions, the concentration of oxygenated HEME in the cell increases, which binds to the oxygen-dependent activator HAP and the HAP-HEME complex promotes the expression of aerobic genes including ROX1. In addition, ROX1 suppresses the expression of anaerobic genes such as ANB1, which are genes further downstream in the oxygen response pathway. In other words, ROX1 functions primarily as a repressor of anaerobic genes under aerobic conditions. At the same time, Rox1 is known to have an autoinhibitory function, as there are ROX1 binding sites upstream of the Rox1 gene itself. As described, there are many promoters in yeast that are regulated by factors dependent on oxygen concentration [14][15][19][26][27].

Fig. 3 Overview of oxygen sensing system in S. cerevisiae.

Some of the promoters repressed by ROX1 have been reported to exhibit anaerobic repressive behavior under plasmid conditions, even though they normally exhibit aerobic repressive behavior in the genome [8][9][10]. In our development, we focus on two of these promoters, pAnb1 and pRox1, and by incorporating fluorescent protein mCherry downstream, we construct the following plasmid and develop a gene circuit that emits fluorescence under aerobic conditions.
In the future, we will incorporate chromoprotein [22], and other transcription factors into downstream of these promoters to create a highly visible, threshold-controlled oxygen reporter.

Fig. 4 Gene design of pRox1 oxygen reporter.

Fig. 5 Oxygen sensing pathway in our system.

Defensin secretion

Humans use various mechanisms as a biological defense system, and one of them is the secretion of antimicrobial peptides. Defensins are the typical family of antimicrobial peptides and many types are known in humans. We chose HBD3 (Human Beta Defensin 3) for this project because it has many advantages over other defensins, such as no decrease in antibacterial activity with salt concentration, and is effective against both gram-positive and gram-negative bacteria [12].
We construct a system for efficient extracellular secretion of defensins by fusing a secretion signal MFα1tag and a constant promoter pCYC, to the upstream of the HBD3 sequence [20].

Fig. 6 Gene design of HBD3 secretion.

Pathogen detection

Many bacteria, including P. aeruginosa, use a chemical substance called AHL (N-acyl-homoserine lactones) for intercellular communication called quorum sensing, which particularly plays a major role in biofilm formation [7]. By detecting these AHLs, P. aeruginosa infection in wounds can be monitored.
The structure of AHLs varies depending on the type of bacteria, but the AHL that plays a major role in P. aeruginosa is 3OC12-HSL [7][13]. Protein LasR and QscR derived from P. aeruginosa are known as the receptors which respond to 3OC12-HSL. These receptors act as activators by forming complexes with AHLs and promote gene transcription [11][25].

In our project, we focus on these two receptors from P. aeruginosa and aim to construct a system to detect the growth of P. aeruginosa by transferring this mechanism to S. cerevisiae, a eukaryotic organism. This is based on a project by the Tsinghua University iGEM team in 2013 [23]. In this system, we need to transfer the prokaryotic system to the eukaryotic one. The VP16 repeats functions as an activation domain (AD) and binds to RNA POL II, and the nuclear localization sequence (NLS) functions as a transcriptional activator by incorporating LasR into the nucleus. To reconstruct the Las promoter in yeast, we also added the cyc100 mini-promoter (TATA box), which functions to recruit RNA POL II to the gene DNA.
With this system,when the concentration of AHL increases, LasR forms a complex with AHL , which binds to the Las promoter, causing the VP16 region to induce RNA POL II and start transcription from the cyc100 promoter. In this way, we can construct a system that can promote the expression of fluorescent protein mCherry when the yeast detects AHL. A similar system is also constructed for QscR.

Fig. 7 Pathogen detection pathway in our system.

While the Tsinghua University 2013 project focused on the LuxR from marine bacteria to build this system, our project will extend and improve it to the LasR and QscR from P. aeruginosa, making it possible to respond to more types of AHLs. This will show the possibility of constructing a broad system that can be used to detect bacteria other than P. aeruginosa, as supported by the fact that QscR and LasR are responsive to AHLs other than 3OC12-HSL [16][24].

Fig. 8 Responsiveness of AHL receptors from P. aeruginosa.(cited from [24])

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