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

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.

Functional Natural Killer-cell Genetics and Microvascular Inflammation After Kidney Transplantation: An Observational Cohort Study.

BACKGROUND: Recent evidence highlights the pivotal role of natural killer (NK) cells in allograft rejection. METHODS: We explored associations of missing self and gene polymorphisms determining the phenotype and/or functionality of NK cells with microvascular inflammation (MVI) in a single-center cohort of 507 consecutive kidney transplant recipients. Patients were genotyped for killer cell Ig-like receptors and polymorphisms in 4 selected genes (FCGR3AV/F158 [rs396991], KLRC2wt/del, KLRK1HNK/LNK [rs1049174], and rs9916629-C/T). RESULTS: MVI was detected in 69 patients (13.6%). In a proportional odds model, the KLRC2del/del variant reduced MVI risk (odds ratio [OR] 0.26; 95% confidence interval [CI], 0.05-0.93; P = 0.037) independent of donor-specific antibodies, HLA class II eplet mismatch, and number of biopsies. Conversely, missing self (OR 1.40; 95% CI, 1.08-1.80; P = 0.011) and the rs9916629 T/T gene variant increased the risk (OR 1.70; 95% CI, 1.08-2.68; P = 0.021). Graft loss tended to be more frequent among patients with missing self ≥2 (hazard ratio 1.97; 95% CI, 0.89-4.37; P = 0.097), without influence on estimated glomerular filtration trajectories. FCGR3A variants were associated with MVI only in patients with preformed and/or de novo donor-specific antibodies (OR 4.14; 95% CI, 0.99-17.47; P = 0.052). CONCLUSIONS: Missing self and NK-cell genetics may contribute to MVI, underscoring the important role of NK cells in transplant rejection.

Streamlined assembly of cloning and genome editing vectors for genus Clostridium.

Reported herein is a new set of vectors designed to streamline molecular cloning and genome editing by exploiting modern cloning methods. The new vectors build on the existing pMTL8000 vectors that have been a staple of Clostridium research for more than a decade. The introduction of two pairs of type IIS restriction sites flanking an insulated multiple cloning site in both a cloning vector and a CRISPR-Cas9 gene editing vector enables plasmid construction in a "one-pot" reaction, avoiding the more laborious steps of conventional cloning. A synthetic lacZα expression cassette introduced between the cloning sites enables visual detection of background colonies. In addition, distinct selection markers on each vector permit selection of the desired clones according to antibiotic resistance. An example of strain development using the new vectors is demonstrated.

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

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.