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
Received: 27 June 2022/Accepted: 15 December 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 a 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
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*Corresponding
author; email: bazliramzi@ukm.edu.my
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