A Clostridioides difficile cell-free gene expression system for prototyping and gene expression analysis

Abstract

Clostridioides difficile is an obligate anaerobic, Gram-positive bacterium that produces toxins. Despite technological progress, conducting gene expression analysis of C. difficile under different conditions continues to be labor-intensive. Therefore, there is a demand for simplified tools to investigate the transcriptional and translational regulation of C. difficile. The cell-free gene expression (CFE) system has demonstrated utility in various applications, including prototyping, protein production, and in vitro screening. In this study, we developed a C. difficile CFE system capable of in vitro transcription and translation (TX-TL) in the presence of oxygen. Through optimization of cell extract preparation and reaction systems, we increased the protein yield significantly. Furthermore, our observations indicated that this system exhibited higher protein yield using linear DNA templates than circular plasmids for in vitro expression. The prototyping capability of the C. difficile CFE system was assessed using a series of synthetic Clostridium promoters, demonstrating a good correlation between in vivo and in vitro expression. Additionally, we tested the expression of tcdB and tcdR from clinically relevant C. difficile strains using the CFE system, confirming higher toxin expression of the hypervirulent strain R20291. We believe that the CFE system can not only serve as a platform for in vitro protein synthesis and genetic part prototyping but also has the potential to be a simplified model for studying metabolic regulations in Clostridioides difficile.IMPORTANCEClostridioides difficile has been listed as an urgent threat due to its antibiotic resistance, and it is crucial to conduct gene expression analysis to understand gene functionality. However, this task can be challenging, given the need to maintain the bacterium in an anaerobic environment and the inefficiency of introducing genetic material into C. difficile cells. Conversely, the C. difficile cell-free gene expression (CFE) system enables in vitro transcription and translation in the presence of oxygen within just half an hour. Furthermore, the composition of the CFE system is adaptable, permitting the addition or removal of elements, regulatory proteins for example, during the reaction. As a result, this system could potentially offer an efficient and accessible approach to accelerate the study of gene expression and function in Clostridioides difficile.

Keywords: CFE system; Clostridioides difficile; in vitro expression; prototyping; tcdR and tcdB expression.

Fig 1

Establishment of the Clostridioides difficile cell-free expression system. (A) A simplified diagram outlining the preparation of the CFE reaction. C. difficile cells were anaerobically inoculated in BHIS medium, then subjected to centrifugation, sonication, and necessary processes to produce the extract. This extract was then supplemented with the energy buffer, amino acids, and other necessary components to establish the complete CFE system. Subsequently, the DNA template was introduced into the CFE system to initiate in vitro protein synthesis. (B) Quantification of protein yield at different time points. The luciferase activity was measured at 0, 0.5, 1.0, 1.5, 2.0, and 2.5 hours with the highest luciferase activity observed at 0.5 hour. (P values were calculated by one-way analysis of variance [ANOVA], ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05.)

Fig 2

Optimization of Clostridioides difficile extract preparation and CFE reaction conditions. (A) The extract preparation was optimized by evaluating T7 RNA Ppolymerase concentrations of 0, 1, 15, 25, and 45 U/µL, with the highest luciferase activity observed at 15 U/µL. In addition, the extract was optimized by testing different reaction temperatures (B), OD values (C), and ultrasonic durations (D). The CFE reaction condition was optimized by testing a range of Mg2+ concentrations (E), K+ concentrations (F), AA (amono acid) concentrations (G), extract percentages (H), and plasmid concentrations (I). (pP values were calculated by one-way ANOVA, ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05).

Fig 3

(A) In vitro expression of different DNA forms as templates for CFE reaction. Equal concentrations of DNA templates were examined, including plasmid, PCR product (PCR), PCR followed by PCR cleanup (PCR + PCR clean), PCR followed by gel extraction (PCR + gel extraction), PCR product with protective sequence (Pro-seq PCR), Pro-seq PCR followed by PCR cleanup (Pro-seq PCR + PCR clean), and Pro-seq PCR followed by gel extraction. The greatest luciferase activity was detected in the case of Pro-seq PCR + PCR clean. (B) Comparison of time required to measure gene expression in vitro and in vivo. The preparation of templates for in vitro expression analysis may only take 1 day, as the template could be linear PCR product, bypassing the laborious plasmid construction and sequencing. Moreover, the cultivation and measurement time for in vitro expression were also reduced. The figure was drawn by Figuredraw (https://www.figdraw.com/). (C) The sequences of Clostridium promoters for in vitro prototyping (D) and in vivo expression analysis (E). The −35 and −10 regions were indicated above the alignment. The single base variation was red-colored. The patterns of the promoter intensity were comparable for both in vivo and in vitro analyses. (P values were calculated by one-way ANOVA, ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05.)

Fig 4

(A) The promoter and 5′UTR regions of tcdR and tcdB with transcriptional start points indicated by arrows and transcriptional factor binding sites marked by boxes. The phylogenetic tree of the promoter and 5′UTR regions of tcdR (B) and tcdB (C) were generated by MEGA software (https://www.megasoftware.net/). Based on phylogenetic trees, tcdR could be classified into four groups, while tcdB could be classified into three groups. The in vitro expression levels of the promoter and 5′UTR regions of tcdR (D) and tcdB (E) from the representative strains were quantified. The hypervirulent strain R20291 produced significantly more TcdR than AN164 and 630 strains, and significantly more TcdB than AN190 and AN164 strains. The negative control consisted of the CFE reaction without a template. (P values were calculated by one-way ANOVA, ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05.)