The Salmonella SehC toxin inhibits growth by cleaving multiple mRNAs
초록/요약
Many bacteria are often resistant to antibiotic treatment and drugs because, even if these drugs are effective, bacteria can slow down their growth rate and thus attenuate the effectiveness of the drug. A similar growth-rate control is detected in pathogenic bacteria that infect and persist inside their hosts. The bacterial growth rate within host cells can be regulated by multiple signaling pathways, most of which are still unknown. A toxin-antitoxin (TA) system is one of the candidates for controlling bacterial growth because the TA system could slow down growth by expressing a toxin component. The toxin protein can be neutralized by the antitoxin component, serving as a non-heritable phenotypic switch for growth rate. In this study, we investigated the SehCD type II TA system from the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium. When the SehC toxin was overexpressed by ectopic production or chromosomal sehD antitoxin was deleted, Salmonella growth was arrested, suggesting that the SehC is a bona fide toxin. We identified that K47, R62, Y65, and Y80 amino acid residues of SehC, which were included in β-sheet1, 2, and 3 were critical for toxin activity. Furthermore, their amino acid substitutions affected Salmonella’s invasion and replication in the macrophage. Salmonella overexpressed SehC could be recovered over time unlike ParE toxin, known as DNA-targeting gyrase inhibitor, and thus we considered SehC toxin as ribonuclease. We analyzed SehC-targeting mRNAs or cleavage sites through a 5'-OH RNA sequencing. We identified numerous protein coding mRNAs were induced or repressed by SehC toxin. Some virulence, transport, transcriptional regulator, and translational regulator related genes were induced and some virulence, electron transport chain (ETC), stress-relevant, membrane, and ABC transporter related genes were repressed when SehC toxin was overexpressed. Many coding mRNAs of those genes were degraded when SehC toxin overexpressed, which include IF-2 translation initiation factor, EF-G translation elongation factor, Rho transcription termination factor, GreA transcription elongation factor, and RplL 50S ribosomal protein L7/L12. Through northern blot analysis, we identified that those mRNAs were very unstable and actually cleaved when SehC toxin overexpressed. However, SehC toxin could not degrade RNA alone in vitro. Therefore, we considered that SehC toxin induces Salmonella growth arrest, possibly by RNase-mediated degradation of these genes. RNase E is the best candidate, because it is essential for mRNA decay and its cleavage sites were also distributed around where we found by SehC toxin’s cleavage. Understanding the underlying mechanism of SehC-mediated mRNA cleavage and growth rate control within host cells will provide a new alternative to treat antibiotic resistant bacteria or intracellular bacteria surviving within host cells.
more초록/요약
Salmonella enterica serovar typhimurium is a gram-negative pathogenic bacteria. Salmonella cause diseases such as typhoid fever, paratyphoid fever, and food poisoning. It is difficult to kill Salmonella because of their unique lifestyle. Salmonella can be colonized in macrophages of host cells with many survival mechanisms. One of their strategies is toxin-antitoxin systems. In this study, we investigated four type II TA systems, parDE, relEB, sehAB, and sehCD from the intracellular bacterial pathogen S. Typhimurium, which are aligned together and formed operon structure. We predicted that parE, relE, sehA, and sehC were toxin and parD, relB, sehB, and sehD were antitoxin. When each of the toxin proteins except for relE was over-expressed, there was a change in the growth of Salmonella. However, over-expressed antitoxin or toxin-antitoxin were not. When antitoxin proteins’ activity is low, toxin proteins can inhibit cell growth. Otherwise, toxin proteins are neutralized by forming a complex with antitoxin proteins. In conclusion, overexpressed sehC gene was a severe growth-arrest among them. Furthermore, overexpressed SehC toxin, the same recombinant plasmid, in E. coli MG1655 showed the same phenotype as in the Salmonella.
more초록/요약
Bacterial growth rate within host cells can be regulated by multiple signaling pathways, most of which are still unknown. Toxin-antitoxin (TA) system is one of the candidates for controlling bacterial growth because TA system could slow down growth by expressing the toxin component. There are many ways to influence the growth by toxin. In this study, we compared to ParE toxin, which is known as DNA-targeting gyrase inhibitor. Therefore, when we overexpressed ParE toxin in Salmonella, it couldn’t be recovered over time. Unlike ParE toxin, Salmonella overexpressed SehC could be recovered over time, and thus we considered SehC toxin as ribonuclease. The toxin protein can be neutralized by the antitoxin component, serving as a non-heritable phenotypic switch for growth rate (Figure 1A). In this study, we investigate SehCD type II TA system from the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium. When SehC toxin is overexpressed or SehD antitoxin is deleted, Salmonella growth is arrested, suggesting that SehC is a bona fide toxin. We identified that K47, R62, Y65, and Y80 amino acid residues of SehC, which were included in β-sheet1, 2, and 3 were critical for toxin activity. Furthermore, their amino acid substitutions affected Salmonella’s invasion and replication in the macrophage. Understanding the underlying mechanism of SehC-mediated growth rate control within host cells will provide a new alternative to treat antibiotic resistant bacteria or intracellular bacteria surviving within host cells. This study suggests a new path that the sterilization of bacteria or the life history can be regulated.
more초록/요약
In this study, we investigated the SehCD type II TA system from the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium. When the SehC toxin was overexpressed by ectopic production, Salmonella growth was arrested, suggesting that the SehC is a bona fide toxin. We considered the SehC toxin as ribonuclease and SehC-targeting mRNAs or cleavage sites were determined by a 5'-OH RNA sequencing. We identified numerous protein coding mRNAs were induced or repressed by SehC toxin. Some virulence, transport, transcriptional regulator, and translational regulator related genes were induced and some virulence, electron transport chain (ETC), stress-relevant, membrane, and ABC transporter related genes were repressed when SehC toxin was overexpressed. Many coding mRNAs of those genes were degraded when SehC toxin overexpressed, which include IF-2 translation initiation factor, EF-G translation elongation factor, Rho transcription termination factor, GreA transcription elongation factor, and RplL 50S ribosomal protein L7/L12. Through northern blot analysis, we identified that those mRNAs were very unstable and actually cleaved when SehC toxin overexpressed. However, SehC toxin could not degrade RNA alone in vitro. Therefore, we considered that SehC toxin induces Salmonella growth arrest, possibly by RNase-mediated degradation of these genes. RNase E is the best candidate, because it is essential for mRNA decay and its cleavage sites were also distributed around where we found by SehC toxin’s cleavage. Understanding the underlying mechanism of SehC-mediated mRNA cleavage and growth rate control within host cells will provide a new alternative to treat antibiotic resistant bacteria or intracellular bacteria surviving within host cells.
more목차
Contents
THE LIST OF FIGURES ············································ i
THE LIST OF TABLES ············································ iv
ABBREVIATIONS ·················································· vi
ABSTRACT ·························································· vii
PartⅠ. Comparison of toxin’s effect among type II TA systems in Salmonella ··········································· 1
Abstract ······························································· 2
Ⅰ. Introduction ····················································· 3
Ⅱ. Materials and Methods ······································· 6
2.1. Bacterial strains, plasmids, oligodeoxynucleotides, and growth conditions ······················································ 6
2.2. Plasmid construction ············································· 7
2.3. Growth test on solid media or liquid media ················· 8
Ⅲ. Results ···························································· 9
3.1. sehC toxin causes a severe growth inhibition in Salmonella ······························································· 9
3.2. sehD antitoxin represses sehC toxin’s effect ············· 10
Ⅳ. Discussion ······················································ 11
Ⅴ. Figures & Tables ·············································· 13
PartⅡ. The characterization of SehC toxin ··············· 27
Abstract ······························································ 28
Ⅰ. Introduction ···················································· 30
Ⅱ. Materials and Methods ······································ 32
2.1. Bacterial strains, plasmids, oligodeoxynucleotides, and growth conditions ····················································· 32
2.2. Plasmid construction ··········································· 33
2.3. Growth test on liquid media ··································· 34
2.4. Protein structure modeling of SehC toxin ·················· 34
2.5. Construction of strains with chromosomal substitutions in the sehC gene ························································· 35
2.6. Construction of strains with chromosomal deletion of sehC, sehD, and sehCD gene ····································· 36
2.7. Quantitative real time-polymerase chain reaction (qRT PCR) ····································································· 37
2.8. Macrophage survival assay ·································· 38
2.9. Serial dilution plating for determining bacteriostatic or bactericidal activity of SehC toxin ································· 39
Ⅲ. Results ··························································· 40
3.1. SehC induces bacteriostasis ································· 40
3.2. SehD antitoxin controls sehC gene’s expression ········ 41
3.3. K47, R62, Y65, and Y80 of sehC gene are critical residues for toxin activity ························································ 42
3.4 SehC affects invasion and replication in macrophage ·· 43
Ⅳ. Discussion ····················································· 44
Ⅴ. Figures & Tables ·············································· 46
PartⅢ. SehC toxin promotes endoribonuclease-mediated mRNA cleavage ······································ 69
Abstract ······························································ 70
Ⅰ. Introduction ···················································· 72
Ⅱ. Materials and Methods ······································ 75
2.1. Bacterial strains, plasmids, oligodeoxynucleotides, and growth conditions ····················································· 75
2.2. Plasmid construction ··········································· 77
2.3. Growth tests on liquid media ·································· 78
2.4. RNA-seq sample preparation and 5'-OH RNA sequencing
···································································· 79
2.4.1. RNA extraction ······································· 79
2.4.2. RNA-seq sample preparation and sequencing
···································································· 80
2.4.3. Data processing and analysis ···················· 80
2.5. Northern hybridization analysis ······························ 81
2.5.1. RNA extraction ········································ 81
2.5.2. Gel electrophoresis and transfer to membrane
···································································· 82
2.5.3. Probe preparation, hybridization and blot scanning ························································ 83
2.6. Western blot analysis ··········································· 84
2.6.1. Sample preparation ·································· 84
2.6.2. Gel electrophoresis and gel staining ············· 85
2.6.3. Transfer to membrane, antibody incubation, and imaging ························································· 85
2.7. SehC protein purification by Ni2+ affinity column ········· 86
2.8. Protein concentration ·········································· 87
2.9. SehC with RNA degradation assay ························· 88
2.10. Construction of strains with chromosomal deletion of the RNase ··································································· 88
Ⅲ. Results ·························································· 90
3.1. Some mRNAs are up-regulated or down-regulated when SehC toxin is overexpressed ······································· 90
3.2. SehC toxin’s cleavage sites are similar to RNase E ···· 91
3.3 SehC toxin targeting mRNAs are unstable ················· 92
3.4. SehC mediated other ribonucleases for cleaving mRNAs
············································································· 93
Ⅳ. Discussion ······················································ 94
Ⅴ. Figures & Tables ·············································· 96
Ⅵ. References ···················································· 129
국문초록 ···························································· 136
