Small RNA AvrA Regulates IscR to Increase the Stress Tolerances in SmpB Deficiency of Aeromonas veronii

Abstract

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.

Keywords: iron deficiency; iron/sulfur clusters; oxidative; stress; virulence.

Figure 1

Transcriptomic analysis between wild type and smpB knockout. (A) Circos diagram of read distribution and gene expression. From inside, green (fold change of gene expression between ΔsmpB and wild type), blue (mRNA expression of wild type A. veronii by rpkm), red (mRNA expression of ΔsmpB by rpkm), black (reads distribution on the reference genome NZ_CP012504.1). (B) Pathways affected by smpB. The horizontal and vertical axes represented as –log(p) value and the pathway names (p < 0.05). (C) Heat map representing the expressions of type IV pilus assembly, type VI secretion system and outer membrane genes.

Figure 2

SmpB deletion decreases the colonization in host tissue. (A) Survival rate of mice treated by wild type A. veronii. (B) Survival rate of mice treated by ΔsmpB strain. (C) Colonization of wild type and ΔsmpB in spleen, kidney, and liver of ICR mice. (D) Spleen coefficient (mg/g). (E) Kidney coefficient (mg/g). (F) Liver coefficient (mg/g). Error bars were shown as SD. ^**p < 0.01, ^*p < 0.05, following by one-way ANOVA, independent t-tests.

Figure 3

The AvrA downregulation and IscR upregulation mediate the oxidative tolerance of ΔsmpB. (A) Transcriptomic comparisons of 11 sRNA between wild type and ΔsmpB. (B) Transcriptomic comparisons of iscR operon genes between wild type and ΔsmpB. (C) Realtime qPCR detection of iscR operon and AvrA. (D) The growth curves of the derivative A. veronii. (E) The survivals of ΔsmpB and wild type in H₂O₂ concentrations ranging from 0 to 10 mM. ^**p < 0.01, ^*p < 0.05, following by one-way ANOVA, independent t-tests.

Figure 4

AvrA increases the tolerances to oxidative stress and iron deficiency. (A) The secondary structure prediction of AvrA by software RNAfold. (B) Growth curves of wild type, ΔavrA and the complemented strain in iron deficiency treatment. (C) The survivals of wild type, ΔavrA and the complemented strain under H₂O₂ stress in the concentrations ranging from 0 to 10 mM (D) Relative expression of iscR operon at stationary phage in iron deficiency treating with DIP. ^**p < 0.01, following by one-way ANOVA, independent t-tests.

Figure 5

AvrA interacts with iscR mRNA by base pairing. (A) Predicted interaction between AvrA and iscR mRNA by the RNAplex program. (B) Basic architecture of the plasmid constructs. (C,D) The relative florescence was measured at 488 nm excitation and 520 nm emission lights after the corresponding constructs were co-transformed in E. coli strain. ^**p < 0.01, following by one-way ANOVA, independent t-tests.

Figure 6

Regulation of SmpB in virulence regulation. The virulence of A. veronii was affected by the expression of virulent proteins the ability of host colonization and the resistance to adversity. Type VI secretion system and pilus assembly were affected on the transcription and translation level by SmpB regulation directly. The sRNA AvrA functioned the stress resistance directly, which was mediated by SmpB indirectly. The arrows in blue showed the positive regulation, and the line in red showed repression. The symbol “+”, “–” indicated upregulation and downregulation, respectively.