Staphylococcus aureus nt5 gene mutation through CRISPR RNA-guided base editing weakens bacterial virulence and immune evasion

Figures

• 

  Figure 1.. Construction of the nt5C166T gene mutation of S. aureus by a CRISPR RNA editing system. (a) Composition of the base editor pnCasSA-bec system. It mainly includes APOBEC1-dCas9, cap1A and rpsL promoters in the pnCasSA-bec system, which is directed to any genomic locus by the Cas9/sgRNA complex to achieve C/T conversion in positions 4 to 8 of the spacer sequence without homologous recombination. PAM: protospacer adjacent motif. (b) The C166 of the nt5 gene was mutated to T in the edited nucleotide strand in the S. aureus RN4220; thus, the stop codon TAA (in red box) was formed at the 166th base, which led to the end of NT5 protein translation. (c–d) The 166th base of the nt5 gene in S. aureus ATCC 25923 was mutated from C to T, resulting in a stop codon TAA (in red box) that disrupted the function of the NT5 protein. (e) The pnCasSA-BEC-NT5sp plasmid was easily cured from the mutant after base editing. The individual colonies obtained after the curing steps were streaked onto the TSB agar plates in the presence (e, right) or absence (e, left) of Cm to test the effectiveness of curing. S. aureus can grow only on TSB plates, indicating that all bacterial plasmids have been…

• 

  Figure 2.. The nt5 mutation induced bacterial DEGs in S. aureus. (a) The growth curve of S. aureus WT and S. aureus MT showed that the inactivation of the nt5 gene had no influence on bacterial growth. Each point represented the OD of the bacterial suspension measured by three different experimental repeats. (b) The volcano plot of gene expression levels was generated from pairwise comparisons between S. aureus WT and S. aureus MT. Dashed lines represented the applied thresholds (analysis of variance p-value of <0.05 and fold difference of >+ 2.0 or <−2.0). Among the 2,556 genes identified in both strains, 38 genes in the S. aureus MT group were significantly upregulated, and 60 genes were significantly downregulated. (c) GO analysis for the DEGs due to nt5 mutation. The top four activities for the DEGs were involved in membrane function, binding, and nucleotide biosynthesis. (d) KEGG enrichment for the DEGs due to nt5 mutation. DEGs were primarily concentrated in cell community, nucleotide metabolism, and amino acid metabolism. (e, f) The GO and KEGG enrichment plots for DEGs. On the left side, the gene IDs and their corresponding values of log2FC were displayed in order from top to…

• 

  Figure 3.. Construction of the nt5 complementation strain by electroporation. (a) Schematic overview of the pSaR-nt5 plasmid. The nt5 complementation cassette was composed of a native promoter and the nt5 gene. (b) The transformation of nt5 complementation cassette into S. aureus was validated by PCR and sequencing.

• 

  Figure 4.. Bacterial physiological response upon DAP exposure between S. aureus WT, S. aureus MT and S. aureus CT. (a) Bacterial growth was inhibited under 0.5 and 1 μg/mL DAP exposure for 12 h. S. aureus MT was more tolerant to DAP exposure than S. aureus WT and S. aureus CT, indicating that the inactivation of the nt5 gene contributed to DAP resistance. Growth at each time point was measured by the optical density of the bacterial suspension at 600 nm with three biological experiment repeats (n = 3). (b) DAP influence on the growth of S. aureus WT, S. aureus MT, and S. aureus CT was detected in a 96-well plate. The MIC of S. aureus MT was higher than that of WT and S. aureus CT. The red box corresponded to 0.5 MIC of DAP. (c) Bacterial membrane potential was measured by fluorimetry assay (n = 3). S. aureus cultures, including S. aureus WT, S. aureus MT, and S. aureus CT were incubated with or without DAP for 5 h to detect membrane potential respectively with 3 times (n = 3). The increase of fluorescence intensity meant reduction of bacterial membrane potential. Under the same DAP concentration, the change in membrane potential of S. aureus MT was smaller than that of S. aureus WT and S.…

• 

  Figure 5.. Silence of nt5 weakened mouse kidney abscess formation. (a) Compared with the normal mouse, the body weight of the mouse was obviously decreased under injection of S. aureus MT, S. aureus WT, and S. aureus CT after injection for 2–4 days. Normal group: normal C57BL/6JGpt mice injected with PBS. (b) The injection of S. aureus MT, S. aureus WT, and S. aureus CT induced mouse kidney abscesses. Yellow arrows indicated abscess lesions and the number of kidneys producing abscess was indicated in brackets. Normal group without kidney abscess: normal C57BL/6JGpt mice injected with PBS. (c) The surviving S. aureus in mouse peripheral blood. We examined the bacterial quantity of surviving in mouse peripheral blood after injection of S. aureus WTand S. aureus MT to C57BL/6JGpt mice for 7 days. (d) The ratio of mouse kidney weight versus mouse body weight under injection of S. aureus MT, S. aureus WT, and S. aureus CT. Normal group: normal C57BL/6JGpt mice injected with PBS. (e) Bacteria burden in kidneys and livers after intravenous challenge of C57BL/6JGpt mice (n = 6) with 106 CFU of S. aureus, S. aureus was enumerated as log10 CFU per gram of tissue 7 days. The data indicated S. aureus…

• 

  Figure 6.. The S. aureus MT alleviated abscess formation by triggering a weaker inflammatory response. (a–b) Phagocytosis of S. aureus by the mouse macrophage cell line RAW 264.7 (a) and human monocyte THP-1 (b). The survival rate of bacteria was calculated by recording the surviving CFU of S. aureus on TSA. (c–e) The level of IL-1β, IL-18, and IL-6 in mouse serum when the S. aureus-infected sepsis model was constructed for 7 days. (f–h) Changes in transcription level of the nucleic acid metabolic pathway mediated by nt5. The mutation of nt5 gene inhibited the transcription of adk and pyk. (i) Nucleic acid metabolic pathway mediated by nt5. The mutant NT5 produced less adenosine due to the loss of function and the inhibition of adk transcription.

• 

  Figure 7.. Hemolytic activity of S. aureus MT. (a–b) Size of hemolytic ring formed by S. aureus WT, S. aureus MT and S. aureus CT on the blood agar plate. S. aureus WT, S. aureus MT and S. aureus CT meant S. aureus wild-type, nt5 mutation, and nt5 complementation, respectively.

• 

  Figure 8.. Compared to the mock transfection of plasmid pCasSA to S. aureus (A), S. aureus with nt5 gene knocked out by CRISPR/Cas9 could not grow in the Cm-containing TSB agar plate due to the too weak homologous recombination ability in S. aureus to repair bacterial double-stranded DNA breaks (B). Cm: chloramphenicol.