Table_1_Small RNA AvrA Regulates IscR to Increase the Stress Tolerances in SmpB Deficiency of Aeromonas veronii.XLSX

The superbacteria Aeromonas veronii displays not only a strong pathogenicity but also the resistance to nine kinds of antibiotics, resulting in the economic losses and health hazards. Small Protein B (SmpB) plays an important role in protein quality control, virulence, and stress reactions. Transcriptomic data revealed that expressions of the type IV pilus assembly and type VI secretion system (T6SS) proteins were downregulated in SmpB deficiency, indicating that the virulence of A. veronii might be attenuated. Although SmpB deletion decreased colonization in the mouse spleen and liver, LD50 of the smpB mutant was not altered as expected, compared with the wild type. Further, the transcriptomic and quantitative RT-PCR analyses showed that the combination of the downregulated AvrA and the upregulated iron-sulfur protein activator IscR, mediated the oxidative tolerance in smpB deletion. Next a reporter plasmid was constructed in which the promoter of iscR was applied to control the expression of the enhanced green fluorescent protein (eGFP) gene. When the reporter plasmid was co-expressed with the AvrA expression into E. coli, the relative fluorescence intensity was decreased significantly, suggesting that AvrA bound to iscR mRNA by base pairing, which in turn relieved the inhibition of iscR and intensified the downstream iron-sulfur proteins. Collectively, the smpB mutant exhibited an attenuated virulence in mice and enhanced tolerances to oxidative stress. This study demonstrates the complexity of gene regulation networks mediated by sRNA in systems biology, and also reflects the strong adaptability of superbacteria A. veronii in the process of evolution.</p

Data_Sheet_1_Absence of tmRNA Increases the Persistence to Cefotaxime and the Intercellular Accumulation of Metabolite GlcNAc in Aeromonas veronii.pdf

Bacterial persisters are a small proportion of phenotypically heterogeneous variants with the transient capability to survive in high concentrations of antibiotics, causing recurrent infections in both human and aquatic animals. Transfer-messenger RNA (tmRNA), which was encoded by the ssrA gene, was identified as a determinant regulator mediating the persistence to β-lactams in the pathogenic Aeromonas veronii C4. The deletion of tmRNA exhibited the increased ability of persister formation most probably due to the reduction of protein synthesis. Transcriptomic and metabolomic analyses revealed that the absence of tmRNA not only significantly elevated the intercellular levels of metabolite GlcNAc and promoted NaCl osmotic tolerance, but also upregulated the expression of metabolic genes in both the upstream biosynthesis pathway and the downstream metabolic flux of peptidoglycan (PG) biosynthesis. Finally, exogenous GlcNAc stimulated significant bacterial growth, enhanced content of GlcNAc in the cell wall, higher resistance to osmotic response, and higher persistence to cefotaxime in a concentration-dependent manner, implying its potential role in promoting the multiple phenotypes observed in tmRNA deletion strains. Taken together, these results hint at a potential mechanism of persister formation mediated by tmRNA against the β-lactam challenges in A. veronii.</p

Table_2_Small RNA AvrA Regulates IscR to Increase the Stress Tolerances in SmpB Deficiency of Aeromonas veronii.XLSX

The superbacteria Aeromonas veronii displays not only a strong pathogenicity but also the resistance to nine kinds of antibiotics, resulting in the economic losses and health hazards. Small Protein B (SmpB) plays an important role in protein quality control, virulence, and stress reactions. Transcriptomic data revealed that expressions of the type IV pilus assembly and type VI secretion system (T6SS) proteins were downregulated in SmpB deficiency, indicating that the virulence of A. veronii might be attenuated. Although SmpB deletion decreased colonization in the mouse spleen and liver, LD50 of the smpB mutant was not altered as expected, compared with the wild type. Further, the transcriptomic and quantitative RT-PCR analyses showed that the combination of the downregulated AvrA and the upregulated iron-sulfur protein activator IscR, mediated the oxidative tolerance in smpB deletion. Next a reporter plasmid was constructed in which the promoter of iscR was applied to control the expression of the enhanced green fluorescent protein (eGFP) gene. When the reporter plasmid was co-expressed with the AvrA expression into E. coli, the relative fluorescence intensity was decreased significantly, suggesting that AvrA bound to iscR mRNA by base pairing, which in turn relieved the inhibition of iscR and intensified the downstream iron-sulfur proteins. Collectively, the smpB mutant exhibited an attenuated virulence in mice and enhanced tolerances to oxidative stress. This study demonstrates the complexity of gene regulation networks mediated by sRNA in systems biology, and also reflects the strong adaptability of superbacteria A. veronii in the process of evolution.</p

Additional file 1 of Genome-wide DNA N6-methyladenosine in Aeromonas veronii and Helicobacter pylori

Additional file 1. The commands for processing SMRT sequencing data

Data_Sheet_1_Peptide Aptamer PA3 Attenuates the Viability of Aeromonas veronii by Hindering of Small Protein B-Outer Membrane Protein A Signal Pathway.xlsx

The small protein B (SmpB), previously acting as a ribosome rescue factor for translation quality control, is required for cell viability in bacteria. Here, our study reveals that SmpB possesses new function which regulates the expression of outer membrane protein A (ompA) gene as a transcription factor in Aeromonas veronii. The deletion of SmpB caused the lower transcription expression of ompA by Quantitative Real-Time PCR (qPCR). Electrophoretic mobility shift assay (EMSA) and DNase I Footprinting verified that the SmpB bound at the regions of −46 to −28 bp, −18 to +4 bp, +21 to +31 bp, and +48 to +59 bp of the predicted ompA promoter (PompA). The key sites C52AT was further identified to interact with SmpB when PompA was fused with enhanced green fluorescent protein (EGFP) and co-transformed with SmpB expression vector for the fluorescence detection, and the result was further confirmed in microscale thermophoresis (MST) assays. Besides, the amino acid sites G11S, F26I, and K152 in SmpB were the key sites for binding to PompA. In order to further develop peptide antimicrobial agents, the peptide aptamer PA3 was screened from the peptide aptamer (PA) library by bacterial two-hybrid method. The drug sensitivity test showed that PA3 effectively inhibited the growth of A. veronii. In summary, these results demonstrated that OmpA was a good drug target for A. veronii, which was regulated by the SmpB protein and the selected peptide aptamer PA3 interacted with OmpA protein to disable SmpB-OmpA signal pathway and inhibited A. veronii, suggesting that it could be used as an antimicrobial agent for the prevention and treatment of pathogens.</p

Data_Sheet_2_Peptide Aptamer PA3 Attenuates the Viability of Aeromonas veronii by Hindering of Small Protein B-Outer Membrane Protein A Signal Pathway.docx

The small protein B (SmpB), previously acting as a ribosome rescue factor for translation quality control, is required for cell viability in bacteria. Here, our study reveals that SmpB possesses new function which regulates the expression of outer membrane protein A (ompA) gene as a transcription factor in Aeromonas veronii. The deletion of SmpB caused the lower transcription expression of ompA by Quantitative Real-Time PCR (qPCR). Electrophoretic mobility shift assay (EMSA) and DNase I Footprinting verified that the SmpB bound at the regions of −46 to −28 bp, −18 to +4 bp, +21 to +31 bp, and +48 to +59 bp of the predicted ompA promoter (PompA). The key sites C52AT was further identified to interact with SmpB when PompA was fused with enhanced green fluorescent protein (EGFP) and co-transformed with SmpB expression vector for the fluorescence detection, and the result was further confirmed in microscale thermophoresis (MST) assays. Besides, the amino acid sites G11S, F26I, and K152 in SmpB were the key sites for binding to PompA. In order to further develop peptide antimicrobial agents, the peptide aptamer PA3 was screened from the peptide aptamer (PA) library by bacterial two-hybrid method. The drug sensitivity test showed that PA3 effectively inhibited the growth of A. veronii. In summary, these results demonstrated that OmpA was a good drug target for A. veronii, which was regulated by the SmpB protein and the selected peptide aptamer PA3 interacted with OmpA protein to disable SmpB-OmpA signal pathway and inhibited A. veronii, suggesting that it could be used as an antimicrobial agent for the prevention and treatment of pathogens.</p

Antimicrobial Zeolitic Imidazolate Frameworks with Dual Mechanisms of Action

The horizontal transfer of drug-resistant genes and the formation of biofilm barriers have threatened the therapeutic efficacy of conventional antibiotic drugs. Development of non-antibiotic agents with high delivery efficiency through bacterial biofilms is urgently required. A pyrithione (PT)-loading zeolitic imidazolate framework (ZIF-8@PT) is synthesized to destroy biofilms and improve the sensitivity of bacteria to PT. ZIF-8@PT can target and destroy the biofilm as well as the cell membrane, promoting the intracellular delivery of PT and possibly its interaction with SmpB, a protein that could regulate the drug resistance of bacteria. ZIF-8@PT effectively suppresses abdominal infections induced by multiresistant Aeromonas veronii C4 in rodent models without systemic toxicity. ZIF-8@PT promises wide applications in treating infections caused by multidrug-resistant bacteria through a dual mechanism of action