Clostridia as Live Biotherapeutics: Can Modified
            <i>Clostridium</i>
            Species Enhance Disease Treatments?

Kubiak, Alekandra M; de Laak, Jella van; Zhang, Yanchao; Bailey, Tom S; Claessen, Luuk; Hittmeyer, Philip; Vlaswinkel, Carlijn; Mowday, Alexsandra; Dubois, Ludwig J; Theys, Jan

doi:10.2217/fmb-2022-0220

Cited by 2 publications

(4 citation statements)

References 18 publications

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“…Clostridium comprises anaerobic endospore-forming bacteria, some of which are industrially important ( 1 ), while others hold potential as medical vehicles (e.g., Clostridium novyi-NT , Clostridium sporogenes , and Clostridium butyricum ), as demonstrated previously ( 2 – 4 ). To exploit their potential in biotechnological applications, the genetic tools of clostridia have been continually developed and advanced over the years ( 5 ).…”

Section: Introductionmentioning

confidence: 74%

“…The toxicity of hemin in bacteria is multifactorial, and our understanding of it is still incomplete ( 42 , 55 ). In some Clostridium species, hemin oxygenase is involved in enhanced aero-tolerance by degrading hemin to Bv ( 56 ), which could improve the colonization capacity in Clostridium -mediated therapy ( 3 ). In order to avoid the external supply of hemin, we attempted to integrate the hemin biosynthetic pathway into the genome of C. sporogenes using the CRISPR-FnCas12a system (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The substrate for the fluorescence reaction (hemin) occurs naturally and can be obtained from dietary sources and from hemoglobin and Bv, a pigment that produced in the intestine. This is an obvious advantage for the application of genetically engineered clostridia in the medical setting, such as intestinal probiotics and intratumoral living therapeutics ( 3 , 61 , 62 ).…”

Section: Discussionmentioning

confidence: 99%

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Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia

Zhang,

Kubiak,

Bailey

et al. 2023

Microbiol Spectr

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Clostridium species have gained attention in industrial and medical applications, and the development of genetic tools has enabled the advancement of the CRISPR-Cas systems for these bacteria. Compared to the primarily used Cas9 from Streptococcus pyogenes , the utilization of Cas12a (previously known as Cpf1) proteins remains incomplete in clostridia, although they exhibit potential advantages, including T-rich recognition for Clostridium genomes and lower toxicity. In this study, we expanded the CRISPR-Cas tools in clostridia by establishing a CRISPR-Cas12a system with two different cas12a genes ( Ascas12a from Acidaminococcus and Fncas12a from Francisella novicida ). The optimized tetracycline-inducible systems were initially determined by the glucuronidase reporter and were used to drive expression of the cas12a genes and crRNAs. Our results demonstrate that the CRISPR-FnCas12a system offers flexible target selection in clostridia, and a specific folding pattern of the precursor crRNA is important to enable high mutation generation efficiency. By using sacB (encoding levansucrase) as a negative marker for plasmid curing and determining the optimal size of the donor DNA template for gene integration in the CRISPR-FnCas12a system, we achieved highly efficient and rapid genome modification, exemplified by the successful engineering of clostridia ( Clostridium butyricum and Clostridium sporogenes ) to produce near-infrared fluorescence from biliverdin and hemin. IMPORTANCE Continued efforts in developing the CRISPR-Cas systems will further enhance our understanding and utilization of Clostridium species. This study demonstrates the development and application of a genome-engineering tool in two Clostridium strains, Clostridium butyricum and Clostridium sporogenes , which have promising potential as probiotics and oncolytic agents. Particular attention was given to the folding of precursor crRNA and the role of this process in off-target DNA cleavage by Cas12a. The results provide the guidelines necessary for efficient genome engineering using this system in clostridia. Our findings not only expand our fundamental understanding of genome-engineering tools in clostridia but also improve this technology to allow use of its full potential in a plethora of biotechnological applications.

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Section: Introductionmentioning

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia

Zhang,

Kubiak,

Bailey

et al. 2023

Microbiol Spectr

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“…have broadened to encompass the exploration of microbe-host interactions and biomedical applications [ 13 ]. Clostridium oral-spore vaccination (COSV) stands out as a notable concept, capitalizing on the highly resistant endospores and anaerobic germination in the small intestine [ 14 ]. This interest in engineering non-pathogenic clostridia will extend to various disease treatments.…”

Section: Introductionmentioning

confidence: 99%

Multiplex genetic manipulations in Clostridium butyricum and Clostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination

Zhang,

Bailey,

Hittmeyer

et al. 2024

Microb Cell Fact

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Background Clostridium spp. has demonstrated therapeutic potential in cancer treatment through intravenous or intratumoral administration. This approach has expanded to include non-pathogenic clostridia for the treatment of various diseases, underscoring the innovative concept of oral-spore vaccination using clostridia. Recent advancements in the field of synthetic biology have significantly enhanced the development of Clostridium-based bio-therapeutics. These advancements are particularly notable in the areas of efficient protein overexpression and secretion, which are crucial for the feasibility of oral vaccination strategies. Here, we present two examples of genetically engineered Clostridium candidates: one as an oral cancer vaccine and the other as an antiviral oral vaccine against SARS-CoV-2. Results Using five validated promoters and a signal peptide derived from Clostridium sporogenes, a series of full-length NY-ESO-1/CTAG1, a promising cancer vaccine candidate, expression vectors were constructed and transformed into C. sporogenes and Clostridium butyricum. Western blotting analysis confirmed efficient expression and secretion of NY-ESO-1 in clostridia, with specific promoters leading to enhanced detection signals. Additionally, the fusion of a reported bacterial adjuvant to NY-ESO-1 for improved immune recognition led to the cloning difficulties in E. coli. The use of an AUU start codon successfully mitigated potential toxicity issues in E. coli, enabling the secretion of recombinant proteins in C. sporogenes and C. butyricum. We further demonstrate the successful replacement of PyrE loci with high-expression cassettes carrying NY-ESO-1 and adjuvant-fused NY-ESO-1, achieving plasmid-free clostridia capable of secreting the antigens. Lastly, the study successfully extends its multiplex genetic manipulations to engineer clostridia for the secretion of SARS-CoV-2-related Spike_S1 antigens. Conclusions This study successfully demonstrated that C. butyricum and C. sporogenes can produce the two recombinant antigen proteins (NY-ESO-1 and SARS-CoV-2-related Spike_S1 antigens) through genetic manipulations, utilizing the AUU start codon. This approach overcomes challenges in cloning difficult proteins in E. coli. These findings underscore the feasibility of harnessing commensal clostridia for antigen protein secretion, emphasizing the applicability of non-canonical translation initiation across diverse species with broad implications for medical or industrial biotechnology.

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Clostridia as Live Biotherapeutics: Can Modified Clostridium Species Enhance Disease Treatments?

Cited by 2 publications

References 18 publications

Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia

Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia

Multiplex genetic manipulations in Clostridium butyricum and Clostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination

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