Sains
Malaysiana 52(2)(2023): 431-439
http://doi.org/10.17576/jsm-2023-5202-09
Development
of Corynebacterium glutamicum as Staphylococcal-Targeting Chassis via
the Construction of Autoinducing Peptide (AIP)-Responsive Expression System
(Pembangunan Corynebacterium glutamicum sebagai Casis Penyasaran Staphylococcos melalui Pembinaan Sistem Pengekspresian Responsif terhadap Peptida Autoaruhan (AIP))
UMMUL
SYAFIQAH RUSLAN1, NURUL
HANUN AHMAD RASTON2,
NUR AZLINA MOHD SHARIF2, NEOH HUI MIN3, SHEILA NATHAN2 &
AHMAD BAZLI RAMZI1,*
1Institute of Systems Biology
(INBIOSIS), Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul
Ehsan, Malaysia
2Department of Biological Sciences and Biotechnology, Faculty of Science
and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul
Ehsan, Malaysia
3UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan
Malaysia, 56000 Cheras, Kuala Lumpur, Malaysia
Diserahkan: 27 Jun 2022/Diterima: 15 Disember 2022
Abstract
Despite
increasing reports of antimicrobial activities of commensal and non-pathogenic
bacteria such as Corynebacterium spp., previous studies on bioengineered
therapeutics traditionally employed probiotics and food-grade bacteria which
limits further advancements into microbial therapeutics research. In this
study, Corynebacterium glutamicum, a generally recognised as safe (GRAS)
and model bacterium was employed as a new chassis for the development of
bioengineered corynebacterial chassis tailored towards Staphylococcus sp.
via autoinducer peptide (AIP)-based quorum sensing (QS) interactions. To
develop C. glutamicum as a staphylococcal-targeting chassis, the
bacteria were transformed with the pResponse plasmid harboring AIP-responding
accessory regulatory proteins agrAC and red fluorescent protein (RFP)
genes under the control of the PaceA and P3 promoter, respectively,
which was expected to stimulate the production of fluorescence signals in the
presence of AIPs. Fluorescence activity of the C. glutamicum pResponse
strain was compared to control C. glutamicum pRFP strain containing only
the P3-RFP gene without the agrAC gene cassette. Using AIP-I as the
input biomolecule, C. glutamicum pResponse strain fluoresced under
different concentrations of AIP-I whereas no fluorescence was observed in the
control C. glutamicum pRFP strain. When tested with S. aureus culture supernatant, the pResponse strain exhibited increasing fluorescence
over the incubation period with the highest fluorescence signal of 183 relative
fluorescence units (R.F.U) was observed at the 48 h point thereby demonstrating a
functional QS-responsive protein expression system in bioengineered C.
glutamicum. These findings demonstrated the feasibility and promising
potential of developing bioengineered C. glutamicum as a
staphylococcal-responsive and -targeting chassis.
Keywords: AIP
signaling; bioengineered chassis; biological
engineering; Corynebacterium
glutamicum; Staphylococcus aureus; synthetic biology
Abstrak
Walaupun
terdapat pertambahan pelaporan berkenaan aktiviti antimikrob oleh bakteria
komensal dan bukan patogen seperti Corynebacterium spp., kajian
terdahulu dalam penghasilan terapeutik terjurutera biologi secara tradisinya
memfokuskan kepada penggunaan probiotik dan bakteria gred makanan yang
mengekang perkembangan kemajuan dalam kajian terapeutik mikrob. Dalam kajian
ini, Corynebacterium glutamicum iaitu sejenis bakteria model dan
dianggap sebagai bakteria selamat (GRAS), telah digunakan untuk pembangunan
casis baharu Corynebacteria yang bertindak balas dan mengaruh khusus kepada
bakteria spesies Staphylococcus melalui tindak balas isyarat penderiaan
kuorum (QS) berasaskan peptida autoaruhan (AIP). Bagi tujuan ini, bakteria C.
glutamicum telah ditransformasi dengan plasmid pResponse yang mengandungi
gen aksesori kawal atur (agr), agrAC dan protein berpendarfluor merah
(RFP) yang masing-masing di bawah kawalan promoter PaceA dan P3 yang
dijangkakan akan merangsang penghasilan isyarat pendarflour dengan kehadiran
AIP. Aktiviti penghasilan pendarflour
oleh strain pResponse C. glutamicum dibandingkan dengan aktiviti strain
kawalan pRFP C. glutamicum yang hanya mengandungi gen P3-RFP tanpa kaset
gen agrAC. Melalui asai menggunakan sebatian AIP-I, strain pResponse C.
glutamicum menghasilkan isyarat pendafluor namun tidak bagi strain kawalan
pRFP apabila diuji dengan kepekatan berbeza AIP-I. Apabila diuji dengan sampel
supernatan daripada S. aureus, strain pResponse mengeluarkan isyarat
pendarfluor yang berkadaran dan selari dengan tempoh eraman. Bacaan isyarat pendarfluor
tertinggi oleh pResponse adalah 183 unit pendarfluor relatif (R.F.U) pada jam
ke-48 yang menunjukkan bahawa sistem penghasilan protein berasaskan tindak
balas QS AIP-I ini telah berjaya diimplementasikan dalam C. glutamicum.
Hasil penemuan daripada kajian ini telah menunjukkan kebolehlaksanaan dan
potensi besar penggunaan C. glutamicum terjurutera biologi sebagai casis
pengesan dan pensasar bakteria jenis Staphylococcus.
Kata kunci: Biologi sintetik; casis terjurutera biologi; Corynebacterium glutamicum;
isyarat AIP; kejuruteraan biologi; Staphylococcus aureus
RUJUKAN
Bomar, L., Brugger, S.D., Yost, B.H.,
Davies, S.S. & Lemon, K.P. 2016. Corynebacterium accolens releases
antipneumococcal free fatty acids from human nostril and skin surface
triacylglycerols. mBio 7(1): 1-13.
Butrico, C.E. & Cassat, J.E. 2020. Quorum sensing
and toxin production in Staphylococcus aureus osteomyelitis:
Pathogenesis and paradox. Toxins 12(8): 516.
Byrd, A.L., Belkaid, Y. & Segre, J.A. 2018. The
human skin microbiome. Nature Reviews Microbiology 16(3): 143-155.
Charbonneau, M.R., Isabella, V.M., Li, N. & Kurtz,
C.B. 2020. Developing a new class of engineered live bacterial therapeutics to
treat human diseases. Nature Communications 11(1): 1738.
Dodds, D., Bose, J.L., Deng, M.D., Dubé, G.R.,
Grossman, T.H., Kaiser, A., Kulkarni, K., Leger, R., Mootien-Boyd, S., Munivar, A., Oh, J., Pestrak, M., Rajpura,
K., Tikhonov, A.P., Turecek, T., Whitfill, T. & Fey, P.D. 2020. Controlling the growth of the skin commensal Staphylococcus
epidermidis using d-alanine auxotrophy. mSphere 5(3): e00360-20.
Ducarmon, Q.R., Kuijper, E.J. & Olle, B. 2021.
Opportunities and challenges in development of live biotherapeutic products to
fight infections. The Journal of Infectious Diseases 223(3): S283-S289.
Hardy, B.L., Bansal, G., Hewlett, K.H., Arora, A.,
Schaffer, S.D., Kamau, E., Bennett, J.W. & Merrell, D.S. 2020.
Antimicrobial activity of clinically isolated bacterial species against Staphylococcus
aureus. Frontiers in Microbiology 10: 2977.
Hardy, B.L., Dickey, S.W., Plaut, R.D., Riggins, D.P.,
Stibitz, S., Otto, M. & Merrell, D.S. 2019. Corynebacterium
pseudodiphtheriticum exploits Staphylococcus aureus virulence
components in a novel polymicrobial defense strategy. mBio 10(1):
e02491-18.
Inda, M.E., Broset, E., Lu, T.K. & de la
Fuente-Nunez, C. 2019. Emerging frontiers in microbiome engineering. Trends
in Immunology 40(10): 952-973.
Jiang, Y., Qian, F., Yang, J., Liu, Y., Dong, F., Xu,
C., Sun, B., Chen, B., Xu, X. & Li, Y. 2017. CRISPR-Cpf1 assisted genome
editing of Corynebacterium glutamicum. Nature Communications 8:
15179.
Kim, S., Yoon, Y. & Choi, K.H. 2015. Pseudomonas
aeruginosa DesB promotes Staphylococcus aureus growth inhibition in
coculture by controlling the synthesis of HAQs. PLoS ONE 10(7):
e0134624.
Kiryukhina, N.V., Melnikov, V.G., Suvorov, A.V.,
Morozova, Y.A. & Ilyin, V. K. 2013. Use of Corynebacterium
pseudodiphtheriticum for elimination of Staphylococcus aureus from
the nasal cavity in volunteers exposed to abnormal microclimate and altered
gaseous environment. Probiotics and Antimicrobial Proteins 5(4):
233-238.
Le, K.Y. & Otto, M. 2015. Quorum-sensing
regulation in staphylococci - An overview. Frontiers in Microbiology 6:
1174.
Lubkowicz, D., Ho, C.L., Hwang, I.Y., Yew, W.S., Lee,
Y.S. & Chang, M.W. 2018. Reprogramming probiotic Lactobacillus reuteri as a biosensor for Staphylococcus aureus derived AIP-I detection. ACS
Synthetic Biology 7(5): 1229-1237.
Marchand, N. & Collins, C.H. 2013. Peptide-based
communication system enables Escherichia coli to Bacillus megaterium interspecies signaling. Biotechnology and Bioengineering 110(11):
3003-3012.
Mashburn, L.M., Jett, A.M., Akins, D.R. &
Whiteley, M. 2005. Staphylococcus aureus serves as an iron source for Pseudomonas
aeruginosa during in vivo coculture. Journal of bacteriology 187(2): 554-566.
Menberu, M.A., Liu, S., Cooksley, C., Hayes, A.J.,
Psaltis, A.J., Wormald, P.J. & Vreugde, S. 2021. Corynebacterium
accolens has antimicrobial activity against Staphylococcus aureus and
methicillin-resistant S. aureus pathogens isolated from the sinonasal
niche of chronic rhinosinusitis patients. Pathogens 10(2): 207.
Munivar, A.M. & Whitfill, T.M. 2015. Therapeutic treatment of skin
disease with recombinant commensal skin microorganisms. Google Patents.
EP3148569A1 https://patents.google.com/patent/EP3148569A1/en.
Ozdemir, T., Fedorec, A.J.H., Danino, T. & Barnes,
C.P. 2018. Synthetic biology and engineered live biotherapeutics: Toward
increasing system complexity. Cell Systems 7(1): 5-16.
Ramsey, M.M., Freire, M.O., Gabrilska, R.A., Rumbaugh,
K.P. & Lemon, K.P. 2016. Staphylococcus aureus shifts toward
commensalism in response to Corynebacterium species. Frontiers in
Microbiology 7: 1230.
Ramzi, A.B., Ku Bahaudin, K.N.A., Baharum, S.N., Che
Me, M.L., Goh, H.H., Hassan, M. & Mohd Noor, N. 2018. Rapid assembly of
yeast expression cassettes for phenylpropanoid biosynthesis in Saccharomyces
cerevisiae. Sains Malaysiana 47(12): 2969-2974.
Ramzi, A.B., Hyeon, J.E., Kim, S.W., Park, C. &
Han, S.O. 2015. 5-Aminolevulinic acid production in engineered Corynebacterium
glutamicum via C5 biosynthesis pathway. Enzyme and Microbial Technology 81: 1-7.
Rottinghaus, A.G., Amrofell, M.B. & Moon, T.S.
2020. Biosensing in smart engineered probiotics. Biotechnology Journal 15(10): e1900319.
Sanchez, S., Rodríguez-Sanoja, R., Ramos, A. &
Demain, A.L. 2018. Our microbes not only produce antibiotics, they also
overproduce amino acids. The Journal of Antibiotics 71(1): 26-36.
Tan, Y., Shen, J., Si, T., Ho, C.L., Li, Y. & Dai,
L. 2020. Engineered live biotherapeutics: Progress and challenges. Biotechnology
Journal 15: e2000155.
Tham, E.H., Koh, E., Common, J.E.A. & Hwang, I.Y.
2020. Biotherapeutic approaches in atopic dermatitis. Biotechnology Journal 15(10): e1900322.
Van der Rest, M.E., Lange, C. & Molenaar, D. 1999.
A heat shock following electroporation induces highly efficient transformation
of Corynebacterium glutamicum with xenogeneic plasmid DNA. Applied
Microbiology and Biotechnology 52(4): 541-545.
Wang, Q., Zhang, J., Al Makishah, N.H., Sun, X., Wen,
Z., Jiang, Y. & Yang, S. 2021. Advances and perspectives for genome editing
tools of Corynebacterium glutamicum. Frontiers in Microbiology 12: 654058.
Wolf, S., Becker, J., Tsuge, Y., Kawaguchi, H., Kondo,
A., Marienhagen, J., Bott, M., Wendisch, V.F. & Wittmann, C. 2021. Advances
in metabolic engineering of Corynebacterium glutamicum to produce
high-value active ingredients for food, feed, human health, and well-being. Essays
in Biochemistry 65: 197-212.
Zuo, F., Chen, S. & Marcotte, H. 2020. Engineer
probiotic bifidobacteria for food and biomedical applications - Current status
and future prospective. Biotechnology Advances 45: 1-12.
*Pengarang
untuk surat-menyurat; email: bazliramzi@ukm.edu.my
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