Team:UI Indonesia/Description

DESCRIPTION

  1. Project Inspiration
  2. Our Project and Goals
  3. References
Project Inspiration

Indonesia has been fighting against diphtheria ever since the 1980s.1 Introduction of diphtheria tetanus and pertussis vaccine (DTP) in the 1980s had successfully reduced pertussis cases significantly.1 In contrast, diphtheria cases remain stagnant and fluctuate over the year.1 Indonesia Ministry of Health had reported linear increase of total diphtheria cases from 2007-2015.2 Finally in 2017, diphtheria outbreak occurred. Some of the reason why diphtheria outbreak happened was partly due to parents were still taken aback after fake vaccine scandal in 2016. Those fake vaccines ranges from boosters of hepatitis B, diphtheria, tetanus and whooping cough. Even though no cases of illness or fatality had been caused by these fake vaccines, the public was enraged and failed to trust the government in ensuring drug safety. Moreover, as Indonesia is a nation home to largest Muslim population in the world, there have been rumours circulating the public regarding ‘non-halal’ vaccines. It is true that there are some vaccines in Indonesia that contains ‘pork-materials’ such as the MR vaccine. Nevertheless, the highest committee of Muslim organization in Indonesia, basing its decision on Muslim’s law, had allowed the usage of the ‘non-halal’ vaccine. It is not surprising, though, to have some family to still deny vaccinating their kids based on this fact. Indonesia will still have a hard time convincing vaccination to their citizens in few years ahead. We suspect that due to these conditions and previous diphtheria outbreaks, cases of diphtheria will persist in the future.

Corynebacterium diphtheriae has the ability to release a protein called Diphtheria Toxin (DT) (Figure 1). Originally, C. diphtheriae does not produce any toxin and it is a normal flora in human respiratory tract. However, a type of virus called corynephages (tox+phage) carry genes that encode toxin and induces toxicity to C. diphtheriae through phage conversion. DT binds to its solely and only receptor called heparin-binding epidermal growth factor-like growth factor (HB-EGF).3 Once bound, DT is internalized, inhibits protein synthesis and triggers cell death.3 This mechanism helps in the colonization of the bacteria and the characteristic appearance of greyish/whitish patch around the throat.

Figure 1. The mechanism of diphtheria toxin in killing healthy cells

Diphtheria is a serious disease and fatal in 5-10% of cases.4 This fatality is mostly caused by the complication of the toxin that affects tissues and organs distal to site of infection, most commonly is the nervous system (neuritis) and the heart (myocarditis). Myocarditis may occur early and can cause heart failure if not treated properly.5 Moreover, the thickened patch around the throat may spread downwards and cause obstruction of the respiratory tract, the most common fatality in children.5

A fast and reliable diagnosis of diphtheria is also essential in determining the course of disease. According to Indonesia Ministry of Health, antibiotic should be given for 14 days in suspected diphtheria patients.6 On the other hand, common respiratory bacterial infection (e.g. streptococcal throat infection) is given a shorter duration of 10-day course antibiotic.7 Once diphtheria is diagnosed, anti-diphtheria should be given as soon as possible to prevent further toxin complications to other organs. In addition, patient suspected with diphtheria should be kept isolated for at least 48 hour.6 Despite diphtheria exerts prominent clinical sign early diagnosis is often difficult. Reason may include patient would only seek medical advice when the disease has progressed.5 Furthermore, in country where DPT vaccine is used widely, cases of diphtheria are rare and diagnosis can be missed as it possess similar early symptoms with upper respiratory tract infection (URTI).5 If false action is to be performed, complication will occur and the contagiousness of diphtheria may cause an outbreak to happen. Moreover, treatment success rate reduces once the disease has progressed. When diphtheria toxin has spread and bound to body tissue, administration of diphtheria antitoxin will not be as effective.

Now, we understand that the diagnosis of diphtheria should be as soon as possible. In Indonesia, ironically, the most widely and commonly used diagnosis is bacterial culture which may take at least 24-48 hour of incubation.6 There are other alternatives including searching the diphtheria DNA by using polymerase chain reaction (PCR), enzyme immunoassay (EIA) and immunochromatographic strip test method (ICT). Unfortunately, despite these methods are quite specific and fast, they are also fairly expensive to be used commercially, especially if it is applied in rural areas all over Indonesia.8

Therefore, our proposed project focuses on improving the weaknesses of current diphtheria diagnostic tools and make it possible as a functional diagnostic tools highly suitable to be used in Indonesia or other developing countries. In order to help Indonesia government in achieving fast and reliable diphtheria diagnostic tools, our project aims to create Escherichia coli that can identify diphtheria toxin. The E. coli that was previously transformed with fluorescent protein, upon binding of diphtheria toxin, will activate the protein and exhibits color when exposed to light. In the near future once our research come to success, we hope that this project can be utilized as primary diphtheria diagnostic tools.

Our Project and Goals

We try to create a chimeric protein between HB-EGF with a protein that regulates E. coli transcription factor and activates our desired fluorescent protein. We then selected EnvZ/OmpR, a two-component regulatory system (TCS) that regulates expression of outer membrane porins at the transcription level (Figure 2).9 EnvZ is a histidine kinase/phosphatase attached on E. coli inner membrane.10 While, OmpR is a transcription factor that regulates the expression of outer membrane porins, particularly the OmpC and OmpF, through the regulatory sequence of ompC and ompF promoters.10

Figure 2. EnvZ/OmpR regulation of porin gene (OmpF and OmpC).13


During low osmolality, EnvZ phosphatase activity is higher than its kinase’s to maintain OmpR high concentration. This will cause OmpF expression to increase. In contrary, high osmolality keeps EnvZ kinase activity to increase than its phosphate’s creating high phosphorylated-OmpR concentration, which increases OmpC expression. OmpC has a smaller pore than OmpF and therefore works most effective in high osmolality.11 Both of these proteins (EnvZ and OmpR) work together as the bacterial osmosensor and its subsequent response regulator proteins. Therefore, we hope when a chimeric protein is generated, upon the binding of DT to its HB-EGF receptor will subsequently activate the TCS. Don't worry! we will use inactivated DT, called DiphTox (BBa_K2607000), created by team iGEM18_UI_Indonesia.12

Next, we will establish a plasmid containing fluorescent protein attached with ompC promoter. We will keep the medium in low osmolality so that the basal activity of OmpC protein is decreased. This will also keep the fluorescent protein to its minimal concentration. Once the TCS is activated through DT binding, OmpR activity increases, binds to ompC promoter and induces fluorescent protein expression.

This is a very simple process to detect the presence of DT in the medium. The advantage of this method is its receptor-antigen specific interaction (Table 1). Another advantage is that it only requires a small amount of chimeric E. coli because bacteria are living organisms and able to grow and replicate inevitably (Table 1). By using this method, laboratory all around Indonesia will have unlimited supplies of chimeric E. coli bacteria culture by singly acquiring it. With this vision, our project will inevitably reduce the cost; provide diphtheria diagnostic tools easily, readily, and widely available in Indonesia; and a wilder dream as a primary diagnostic tool for diphtheria detection all around the world.

Table 1. Advantages and disadvantages of our projects
Advantages Disadvantages
Receptor-antigen specific Requires optimum environment for bacteria to strive
Cheap Possible mutation in long term use
High bacteria replication Personnel requires minimal medical science background to operate
References
  1. WHO. Indonesia EPI Fact Sheet [Internet]. 2016 [cited 2019 Jun 14]. Available from: www.depkes.go.id
  2. Pusdatin. Situasi imunisasi di Indonesia [Internet]. Jakarta: Pusat Data dan Informasi Kementerian Kesehatan Republik Indonesia; 2016 [cited 2019 Jun 14]. Available from: http://www.depkes.go.id/resources/download/pusdatin/infodatin/InfoDatin-Imunisasi-2016.pdf
  3. Vinante F, Rigo A. Heparin-binding epidermal growth factor-like growth factor/diphtheria toxin receptor in normal and neoplastic hematopoiesis. Toxins (Basel) [Internet]. 2013 Jun [cited 2019 Jun 16];5(6):1180–201. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23888518
  4. World Health Organization. Diphtheria [Internet]. WHO. World Health Organization; 2018 [cited 2019 Jun 14]. Available from: https://www.who.int/immunization/diseases/diphtheria/en/
  5. CDC. Epidemiology and prevention of vaccine-preventable diseases | CDC [Internet]. 13th ed. Centers for Disease Control and Prevention; 2017 [cited 2019 Jun 18]. Available from: https://www.cdc.gov/vaccines/pubs/pinkbook/index.html
  6. Kemenkes Republik Indonesia. Pedoman pencegahan dan pengendalian difteri [Internet]. Jakarta; 2017 [cited 2019 Jun 19]. Available from: http://sehatnegeriku.kemkes.go.id/wp-content/uploads/2018/01/buku-pedoman-pencegahan-dan-penanggulangan-difteri.pdf
  7. Altamimi S, Khalil A, Khalaiwi KA, Milner RA, Pusic M V, Al Othman MA. Short-term late-generation antibiotics versus longer term penicillin for acute streptococcal pharyngitis in children. Cochrane Database Syst Rev [Internet]. 2012 Aug 15 [cited 2019 Jun 19]; Available from: http://doi.wiley.com/10.1002/14651858.CD004872.pub3
  8. PHLN. Diphtheria lLaboratory Case Definition (LCD) [Internet]. Australian Government Department of Health and Ageing; 2009 [cited 2019 Jun 23]. Available from: https://www.health.gov.au/internet/main/publishing.nsf/Content/cda-phlncd-diphtheria.htm
  9. Wang LC, Morgan LK, Godakumbura P, Kenney LJ, Anand GS. The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm. EMBO J [Internet]. 2012 [cited 2019 Jun 16];31(11):2648. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365433/
  10. Flores-Valdez MA, Ferná Ndez-Mora M, Ngel Ares MA´, Giró JA, Calva E, Ngel De La Cruz MA´. OmpR phosphorylation regulates ompS1 expression by differentially controlling the use of promoters. Microbiology [Internet]. 2014 [cited 2019 Jun 17];160:733–41. Available from: http://mic.sgmjournals.org
  11. Yoshida T, Qin L, Egger LA, Inouye M. Transcription regulation of ompF and ompC by a single transcription factor, OmpR. J Biol Chem [Internet]. 2006 Jun 23 [cited 2019 Jun 18];281(25):17114–23. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16618701
  12. Laurentius A. Part:BBa K2607000 [Internet]. 2018 [cited 2019 Jun 18]. Available from: http://parts.igem.org/Part:BBa_K2607000
  13. Feng X, Oropeza RS, Walthers D, Kenney LJ. OmpR Phosphorylation and Its Role in Signaling and Pathogenesis This two-component system response regulator modulates multiple systems, including virulence factors and fimbriae [Internet]. 2003 [cited 2019 Jun 18]. Available from: https://www.semanticscholar.org/paper/OmpR-Phosphorylation-and-Its-Role-in-Signaling-and-Feng-Oropeza/82b6f7c28587f0cc69828986a588c5e6d121d29d