Analysis of essential genes in Clostridioides difficile by CRISPRi and Tn-seq

Abstract

Essential genes are interesting in their own right and as potential antibiotic targets. To date, only one report has identified essential genes on a genome-wide scale in Clostridioides difficile, a problematic pathogen for which treatment options are limited. That foundational study used large-scale transposon mutagenesis to identify 404 protein-encoding genes as likely to be essential for vegetative growth of the epidemic strain R20291. Here, we revisit the essential genes of strain R20291 using a combination of CRISPR interference (CRISPRi) and transposon insertion site sequencing (Tn-seq). First, we targeted 181 of the 404 putatively essential genes with CRISPRi. We confirmed essentiality for >90% of the targeted genes and observed morphological defects for >80% of them. Second, we conducted a new Tn-seq analysis, which identified 346 genes as essential, of which 283 are in common with the previous report and might be considered a provisional essential gene set that minimizes false positives. We compare the list of essential genes to those of other bacteria, especially Bacillus subtilis, highlighting some noteworthy differences. Finally, we used fusions to red fluorescent protein (RFP) to identify 18 putative new cell division proteins, 3 of which are conserved in Bacillota but of largely unknown function. Collectively, our findings provide new tools and insights that advance our understanding of C. difficile.IMPORTANCEClostridioides difficile is an opportunistic pathogen for which better antibiotics are sorely needed. Most antibiotics target pathways that are essential for viability. Here, we use saturation transposon mutagenesis and gene silencing with CRISPR interference to identify and characterize genes required for growth on laboratory media. Comparison to the model organism Bacillus subtilis revealed many similarities and a few striking differences that warrant further study and may include opportunities for developing antibiotics that kill C. difficile without decimating the healthy microbiota needed to keep C. difficile in check.

Keywords: CRISPRi; Tn-seq.

Fig 1

Summary of gene essentiality determined by CRISPRi. (A) Representative spot titer assays illustrating the range of viability defects observed. Strains of R20291 harboring CRISPRi plasmids were grown overnight in TY-Thi10 medium. Samples were serially diluted in 10-fold increments, and 5 µL of each dilution was spotted onto TY-Thi10 plates with 1% xylose to induce the expression of dCas9. Plates were photographed after incubation overnight. (B) Scoring of CRISPRi screen. Top: Criteria for assigning viability defects based on colony size and the highest dilution at which growth was observed. Bottom: 92% of genes tested showed a viability defect. In cases where the two sgRNAs produced different results, the stronger viability defect was used. (C) Viability defects in CRISPRi correlate with a likelihood that a gene will be scored as essential by Tn-seq. Viability defects are from Table S1. Tn-seq calls come from Table S3.

Fig 2

Morphology of CRISPRi strains with sgRNAs targeting genes in select functional pathways. Left: pathway. Middle: Predicted transcription unit. Targeted genes are boxed and indicated above the operon diagrams. Numbers are R20291 locus tags. Genes are color-coded to indicate essentiality based on Tn-seq calls in Table S3. Dark blue: essential. Light blue: ambiguous. White: non-essential. Operon structure is not to scale. Right: Morphological changes based on phase contrast and fluorescence micrographs of cells scraped from viability plates. Membranes were stained with FM4-64, and DNA was stained with Hoechst 33342. The size bars are 5 µm. The control strain expressed an sgRNA that does not target anywhere in the genome. Micrographs are representative of at least two experiments. Figure S1 shows microscopy of more genes.

Fig 3

Essentiality follow-up. (A) Transposon insertion profile for murJ2. Vertical lines represent mapped insertion sites and are scaled to indicate the number of sequence reads mapping to that site. Although murJ2 sustained numerous insertions, ~80% were in the last 10% of the gene, suggesting the non-essentiality call by TRANSIT2 is incorrect. (B) Transposon insertion profile for polA indicates that only the N-terminal domain is essential. Read frequency was scaled to 5 to highlight the absence of reads in the N-terminal domain. The average number of reads per polA site with at least one read was 173. (C) Spot titer assays of CRISPRi strains targeting genes in the atp operon. Serial dilutions of overnight cultures were spotted on TY-Thi10 plates with 1% xylose. Plates were imaged after incubation at 37°C for ~18 h. Silencing atpB and atpD resulted in small colonies, while growth after silencing atpI and atpF was comparable to the negative control. (D) Pre-depletion of ATP synthase proteins impairs growth. Starter cultures were grown overnight in TY-Thi10 without (left) or with (right) 1% xylose, then subcultured into TY-Thi10 with 1% xylose, and growth was followed by measuring optical density at 600 nm. To prolong growth, cultures in the left panel were back-diluted at 7 h. (E) Zone of inhibition assays reveal CRISPRi knockdown of the atp operon increases sensitivity to CCCP. Plates were imaged after incubation at 37°C for ~18 h. Novobiocin (Novo) served as a control. Guides in panels C–E were as follows: atpI (5531), atpB (5583), atpF (5581), atpD (5579), or a negative control that does not target anywhere in the genome.

Fig 4

Comparison of Tn-seq data sets using Sankey diagrams. (A) Of the 346 genes classified as essential in this study, 283 were essential, 10 were ambiguous, and 53 were non-essential for Dembek et al. (B) Of the 404 genes classified as essential by Dembek et al., 283 were essential, 35 were ambiguous, and 74 were non-essential in this study. The thickness of each color line is proportional to the number of genes, and labels on the left and right indicate essentiality calls. Short refers to genes that are too short to be called by TRANSIT2. Note that 12 genes identified as essential by Dembek could not be mapped to our data set as they were not present in the genome annotation used here. Category “ambiguous” combines unclear with unclear/NE from Table S3.

Fig 5

Representative fluorescence micrographs of fixed cells that produced the indicated proteins fused to RFP. Percentages indicate the fraction of cells scored positive for septal localization (n ≥ 202 cells). Size bar = 10 µm.