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 RAMZI^1,*

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