Team:UI Indonesia/Description

OVERVIEW

Helicobacter pylori (H. pylori) is a gram negative spiral shaped microaerophilic bacteria that resides in the stomachs of almost half the world population. Its pathogenic effect due to numerous virulence factors lead to various gastric pathologies including peptic ulcer disease and gastric cancer. Although it has been established since 1982, we are facing some treatment challenges especially regarding the biofilm formation and its role in antibiotic resistance. So, we engineer an E. coli, specifically E. coli Nissle 1917, a probiotic strain to be able to (1) chase the H. pylori, (2) disperse its biofilm using a protease: Proteinase-K, and (3) kill the H. pylori using a antimicrobial peptide, PGLa-AM1.

What is Helicobacter pylori?

H. pylori is a pathogenic bacteria that damages gastric mucosa and epithelium.

Why do we concerned?

It is all around us : One of two people has H. pylori inside their stomach which is transmitted oral and fecally. Socioeconomic and demographic play an important role for its existence.


Why do we concerned?

Catastrophic ailments : H. pylori infection results in numerous catastrophic diseases from peptic ulcer to gastric cancer.


Why do we concerned?

Treatment challenges : H. pylori eradication challenges arise from the emerging resistance to antibiotics along with the involvement of biofilm formation.

What is Helicobacter pylori?

Helicobacter pylori (H. pylori) is a gram negative with a spiral shaped bacteria that lives in a microaerophilic environment. This is one of the most commonly found gastrointestinal tract pathogens since its identification in 1982 by Marshall and Warren.1 It is the most well studied bacteria residing in the stomach.




As it reaches the gastric mucosa, it will adhere to gastric epithelium by some adhesion molecules, mostly outer membrane protein. H. pylori has numerous virulence factors, yet there are two most important and well established: cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA) which are secreted through type-4 secretion system (T4SS) lead to gastric mucosal and epithelial damage.

Actually, H. pylori is not an acidophilic bacteria, but it is escaping the acidic environment of the gastric lumen by having urease which is responsible for ammonium production and acts as an acid resistance mechanism. H. pylori then moves like a corkscrew with its spiral like shape and flagella through the gastric mucus from the lumen to the gastric mucosa which is rich for urea as their chemoattractant.2,3


The toxins produced, along with H. pylori pathogen-associated molecular patterns (PAMP) such as lipopolysaccharide (LPS), also activate immune response, leading to inflammation.1-3

Why are we concerned about H. pylori?

It is all around us

Inhabiting approximately half of the world population3,4, this gram-negative bacilli is transmitted by means of oral-oral and fecal-oral, thus can be easily found infecting family members of the infected, suspectedly by sharing feeding utensils.






Even though the COVID-19 pandemic is a new emerging disease which has become a worldwide interest, this does not change the reality that many diseases including H. pylori infection still exist and are not decreasing

Hygiene and socioeconomic status also play a huge factor in H. pylori transmission, therefore a higher prevalence can be observed in developing countries1,4. Based on a preliminary study, the estimation that 22.1% of dyspeptic individuals in Indonesia bear this pathogen. However this numbers may varied between different ethnics (2.4-42.9%).5

It is becoming more important since stomach ache or dyspepsia is one of the most common complaints that brings patients to doctors.

Catastrophic ailments

Persistent H. pylori may cause a plethora of diseases, namely atrophic gastritis and peptic ulcer disease which, in turn, triggers the development of gastric malignancy from adenocarcinoma to lymphoma.1,6,7 For the latter reason, H. pylori is known as a class 1 carcinogen.8



There is no screening for H. pylori nor for gastric cancer in Indonesia. Furthermore, there is an increase up to 40 times the probability to die of gastric carcinoma for those who are positive for H. pylori infection.10

Being the most important risk factor of gastric cancer, it is estimated that 89% of non-cardia gastric cancer cases are attributable to H. pylori infections and 36.3% of all cancer caused by infections around the world in 2008.9

The gastric cancer itself killed 738,000 lives every year and there is a trend of increasing incidence among younger population.11 These facts may result in underdiagnosis and our inability to treat at an earlier stage. Whereas, only less than 12 months for patients with advanced gastric cancer to survive.11

Treatment challenges

The emerging resistance of H.pylori to standard drugs poses a serious problem, arised from abuse of antibiotic use to incomplete eradication. A 2010 systematic review had shown a high global internal resistance of metronidazole, clarithromycin, and levofloxacin (26.7%, 17.2%, and 16.2% respectively).



One of the most underlooked attributable factors to this recalcitrant nature of H. pylori is its production of biofilm, which is largely composed of protein.15,16 It is reported the efficacy of antibiotics decreases many-folds in the culture with biofilm compared to the planktonic culture.2,15 Together with the internal resistance, this combination is a major obstacle for completely treating patients with H. pylori.

The resistance patterns also differ between continents.12 In Indonesia alone, high resistance of metronidazole (49%) and levofloxacin (35%) are observed.13 Not to underestimate the long duration of antibiotic consumption (14 days) which is exhaustive.14

Unfortunately, all previous approaches were ignoring the presence of biofilm. These facts give rise to the interest and urgency to target both the biofilm and find novel antimicrobials to overcome resistance among the bacterium itself.

So, how do we overcome these problems?

Some inspirations

E. coli is a gram-negative bacilli which has been an interest in the field of synthetic biology due to its ability to reproduce in common, readily available medium, thus enabling it to be upscaled to industrial scale.






1. Chemotaxis
Detect H. pylori through quorum sensing molecules, AI-2,21,22 and swim toward.23–25 This approach is also inspired by iGEM ATOMS-Turkiye 2013.26

Due to its manipulable nature, engineered E. coli may serves as the future of biomedical technology, and also be part of the solution for aforementioned problems caused by H. pylori.17

2. Biofilm dispersing
Disperse biofilm with a potent protease, Proteinase-K, to open the H. pylori defense to antibiotic27,28 The secretion of Proteinase-K is done through autolysis of our E. coli by E7 lysis protein.29

One of the most used strains in therapeutic studies is E. coli Nissle 1917 (ECN).18,19 Furthermore, since our system will work in an acidic environment, an acid tolerant bacteria is critical. This property is presence in ECN.20 So, we engineer an ECN that is capable for

3. H. pylori eradication
Kill H. pylori with a specific antimicrobial peptide (AMP), PGLa-AM1, as a new modality with less resistance to overcome the antibiotic resistance.30,31 Again, the release of AMP is done through autolysis in a time dependent manner inspired by the iGEM TU-Delft 2013.32

And at last, we are hoping and working to completely resolve the never ending struggle of H. pylori infection





Figure 1. Helicostrike system overview




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