CRISPR-cas3 of Salmonella Upregulates Bacterial Biofilm Formation and Virulence to Host Cells by Targeting Quorum-Sensing Systems
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains, Plasmids, Primers, and Growth Conditions
2.2. Construction of Cas3 Gene Deletion Strain Δcas3
2.3. Construction of Cas3 Gene Complementary Strain Δcas3/pBAD3-CM-cas3
2.4. Determination of Standard Growth Curve
2.5. Biofilm Assays
2.6. Cell Culture Infection Assays
2.7. Determination of the 50% Lethal Dose (LD50) for SPF Chicken during Salmonella Oral Infection
2.8. Transcriptome Analysis by RNA-Seq
2.8.1. RNA Isolation, Library Construction, Sequencing, and Sequence Data Filtering
2.8.2. Differentially Gene Expression (DEGs), Clustering Analysis, and Functional Analysis of DEGs
2.8.3. Data Validation by RT-qPCR
2.9. Statistical Analysis
3. Results
3.1. Deletion of Cas3 has No Effect on Bacterial Growth
3.2. Cas3 Deletion Impacts Bacterial Biofilm Formation and Cell Infection
3.3. Deletion of Cas3 Increased the LD50 Dose for SPF Chickens during Oral Infection
3.4. Transcriptomics Analysis Reveals Differentially Expressed Genes between Cas3 WT and Δcas3 Strains
3.4.1. GO and KEGG Analysis Identifies Functional Relevance to DEGs
3.4.2. The Patterns of DEGs are Similarly Revealed by RT-qPCR Analysis
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Locus Tag | Gene | Protein | Fold Change (Δcas3/cas3 WT) | p-Value |
---|---|---|---|---|
AV79_RS14235 | cas3 | type I CRISPR-associated protein DNA helicase Cas3 | −301.27 | 5.331 × 10−11 |
AV79_RS14230 | cse1 | type I-E CRISPR-associated protein Cse1 | 20.47 | 2.96 × 10−5 |
AV79_RS14210 | cas6 | type I-E CRISPR-associated protein Cas6 | 9.11 | 6.002 × 10−5 |
AV79_RS14200 | cas2 | CRISPR-associated protein Cas2 | 8.66 | 0.0003026 |
AV79_RS14215 | cas5 | type I-E CRISPR-associated protein Cas5 | 8.34 | 0.0002544 |
AV79_RS14205 | cas1 | CRISPR-associated protein Cas1 | 8.25 | 0.0001503 |
AV79_RS14220 | cas7 | type I-E CRISPR-associated protein Cas7 | 7.68 | 0.0004107 |
AV79_RS14225 | cse2 | type I-E CRISPR-associated protein Cse2 | 6.98 | 0.0006198 |
Gene | Protein | Fold Change (Δcas3/cas3 WT) | p-Value | Function |
---|---|---|---|---|
lsrF | putative aldolase, 3-hydroxy-5-phosphonooxypentane-2, 4-dione thiolase LsrF | 13.48 | 0.01 | Involved in the degradation of phospho-AI-2, thereby terminating induction of the lsr operon and closing the AI-2 signaling cycle. Catalyzes the transfer of an acetyl moiety from 3-hydroxy-5-phosphonooxypentane-2, 4-dione to CoA to form glycerone phosphate and acetyl-CoA. |
lsrG | (4S)-4-hydroxy-5-phosphonooxypentane-2, 3-dione isomerase, autoinducer-2 (AI-2) modifying protein LsrG | 7.90 | 0.02 | Involved in the degradation of phospho-AI-2, thereby terminating induction of the lsr operon and closing the AI-2 signaling cycle. Catalyzes the conversion of (4S)-4-hydroxy-5-phosphonooxypentane-2, 3-dione (P-DPD) to 3-hydroxy-5-phosphonooxypentane-2, 4-dione (P-HPD). |
lsrE | Ribulose-phosphate 3-epimerase, Putative epimerase LsrE | 7.47 | 0.03 | Cofactor. |
lsrB | Autoinducer 2-binding protein LsrB | 6.88 | 0.03 | Part of the ABC transporter complex LsrABCD involved in autoinducer 2 (AI-2) import. Binds AI-2 and delivers it to the LsrC and LsrD permeases. |
lsrA | putative ABC transporter ATP-binding protein, Autoinducer 2 import ATP-binding protein LsrA | −2.60 | 0.15 | Part of the ABC transporter complex LsrABCD involved in autoinducer 2 (AI-2) import. Responsible for energy coupling to the transport system. |
lsrR | transcriptional repressor LsrR | −1.39 | 0.36 | In the absence of autoinducer 2 (AI-2), represses transcription of the lsrACDBFGE operon and its own transcription. In the presence of AI-2, LsrR is inactivated by binding phospho-AI-2, leading to the transcription of the lsr genes |
lsrD | ABC transporter membrane protein, Autoinducer 2 import system permease protein LsrD | 1.55 | 0.38 | Part of the ABC transporter complex LsrABCD involved in autoinducer 2 (AI-2) import. Probably responsible for the translocation of the substrate across the membrane. |
lsrC | sugar transport protein, Autoinducer 2 import system permease protein LsrC | −1.61 | 0.40 | Part of the ABC transporter complex LsrABCD involved in autoinducer 2 (AI-2) import. Probably responsible for the translocation of the substrate across the membrane. |
lsrK | autoinducer-2 (AI-2) kinase | 1.32 | 0.48 | Catalyzes the phosphorylation of autoinducer-2 (AI-2) to phospho-AI-2, which subsequently inactivates the transcriptional regulator LsrR and leads to the transcription of the lsr operon. Phosphorylates the ring-open form of (S)-4, 5-dihydroxypentane-2, 3-dione (DPD), which is the precursor to all AI-2 signaling molecules, at the C5 position. |
luxS | S-ribosylhomocysteinase | 1.36 | 0.40 | Involved in the synthesis of autoinducer 2 (AI-2) which is secreted by bacteria and is used to communicate both the cell density and the metabolic potential of the environment. The regulation of gene expression in response to changes in cell density is called quorum sensing. Catalyzes the transformation of S-ribosylhomocysteine (RHC) to homocysteine (HC) and 4, 5-dihydroxy-2, 3-pentadione (DPD). |
safA | Lipoprotein, Saf-pilin pilus formation protein safA | −3.79 | 0.02 | One of major fimbrial subunits. |
safB | pili assembly chaperone protein SafB | −3.28 | 0.02 | Involved in the organization of pilus and the chaperone-mediated protein folding. |
safC | atypical fimbria outer membrane usher SafC | −2.30 | 0.02 | Involved in pilus assembly and positive to fimbrial usher porin activity. |
safD | fimbrial structural subunit SafD | −2.72 | 0.01 | Part of fimbrial structures. |
bdm | biofilm-dependent modulation protein BDM | 3.49 | 0.04 | Bdm acts as a transcriptional activator for genes that are involved in the flagella formation and was shown to be downregulated in biofilms. |
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Cui, L.; Wang, X.; Huang, D.; Zhao, Y.; Feng, J.; Lu, Q.; Pu, Q.; Wang, Y.; Cheng, G.; Wu, M.; et al. CRISPR-cas3 of Salmonella Upregulates Bacterial Biofilm Formation and Virulence to Host Cells by Targeting Quorum-Sensing Systems. Pathogens 2020, 9, 53. https://doi.org/10.3390/pathogens9010053
Cui L, Wang X, Huang D, Zhao Y, Feng J, Lu Q, Pu Q, Wang Y, Cheng G, Wu M, et al. CRISPR-cas3 of Salmonella Upregulates Bacterial Biofilm Formation and Virulence to Host Cells by Targeting Quorum-Sensing Systems. Pathogens. 2020; 9(1):53. https://doi.org/10.3390/pathogens9010053
Chicago/Turabian StyleCui, Luqing, Xiangru Wang, Deyu Huang, Yue Zhao, Jiawei Feng, Qirong Lu, Qinqin Pu, Yulian Wang, Guyue Cheng, Min Wu, and et al. 2020. "CRISPR-cas3 of Salmonella Upregulates Bacterial Biofilm Formation and Virulence to Host Cells by Targeting Quorum-Sensing Systems" Pathogens 9, no. 1: 53. https://doi.org/10.3390/pathogens9010053