IscR-tmRNA regulatory axis plays a key role in multiple stress response and pathogenicity in Aeromonas veronii

Abstract

Bacterial pathogens intricately modulate their response to a variety of stress and the virulence, particularly in light of the dynamic conditions both in natural habitat and within host organisms. Transfer-messenger RNA (tmRNA), which plays an important role in pathogenicity due to its major function in the trans-translation system for ribosome rescue, has been proved as a stress response molecule. Herein, our results indicate that the global regulator IscR acts as a crucial activator responsible for the expression of tmRNA in Aeromonas veronii, a bacterial pathogen posing significant challenges to both aquatic industry and public health. Bacterial one-hybrid and electrophoretic mobility shift assays (EMSA) confirm the direct binding of IscR to the promoter region of the ssrA gene which encodes tmRNA. Moreover, our phenotypic characterizations illustrate that the complementation of tmRNA can rescue the defects of iscR deletion in response to adverse stress, including nutrient deprivation, elevated temperatures, β-lactam antibiotics, and oxidative stress, as well as in establishing the pathogenicity characterized by motility, aggregation, adhesion, cytotoxicity, bacterial competition, and colonization in mice. Our findings offer insights into a potential model for strengthening bacterial survival in external environments, and provide an initial glimpse into the intricate interplay between the functional roles of IscR and tmRNA in the pathogenicity through the IscR-tmRNA regulatory axis.

Keywords: Aeromonas veronii; IscR; pathogenicity; stress response; tmRNA; virulence.

Figure 1.

IscR activates the expression of tmRNA by binding to the promoter region of ssrA gene. (a) RT-qPCR detections of the mRNA levels of iscR, ssrA, and smpB genes in ΔiscR and its complementation strains of A. veronii C4. cDNA was synthesized using 1 μg total RNA as the template and subsequently diluted for RT-qPCR reaction. The relative expression levels were quantified by 2^−ΔΔCT method using 16S rRNA as a reference for data normalization. (b) egfp reporter gene assay by co-transforming E. coli XL 1-Blue MRF’reporter strain with the transcriptional fusion pBBR-PssrA-eGFP and the expression vector pTRG-IscR or pTRG. After incubation overnight, aliquots of 2 x 10⁸ cells were harvested at a 3-h interval for the measurement of eGFP fluorescence intensity, and the transcriptional activity is shown in terms of eGFP fluorescence values normalized to OD₆₀₀. (c) Bacterial one-hybrid assay with a reporter strain expressing ssrA promoter (upper panels) or its mutants (lower panels) together with full length IscR. Strains containing pBX-LGF2/pTRG-Gal11^p and the empty vector pBXcmT/pTRG were used as the positive and negative control strains, respectively. The co-transformants were selected on the His-selective plates (+3at, +Str^r). (d) EMSA analysis performed with 40 ng 6-FAM labeled 169-bp promoter region of ssrA gene (the upper panel) or its mutants (the lower panel) together with different quantities of purified IscR protein. After incubation in the binding reaction system for 30 min at 30°C, the complex was visualized by electrophoresis and subsequent detection of the fluorescence. (e) Diagrammatic representation showing E. coli type 2 IscR box and the putative IscR binding sequences in the upstream region of ssrA promoter region. The sequences marked green in the putative binding sites were mutated as shown in red to obtain the mutants PssrA-1, and PssrA-2, respectively. Error bars in (A) and (B) represent standard deviations from triplicate experiments. Tukey’s post-test was used for statistical analysis, with * representing p < 0.05, ** representing p < 0.01, *** representing p < 0.005, and ns indicating no significant difference.

Figure 2.

IscR-tmRNA regulatory axis mediates the responses to poor nutrition, temperature, β-lactam antibiotic, and oxidative stress. Bacterial growth under various challenge conditions was recorded by measuring OD₆₀₀ at 1-h intervals and the growth curves were plotted by the values of OD₆₀₀ versus the incubation time. The challenged conditions are as follows: (a) Reduced oxygen under static culture and limited nutrition in M9 minimal medium; (b) Temperature stress under 22°C, 30°C or 42°C; (c) β-lactam antibiotic stress under 0.1, 0.2, or 0.3 ug/ml of cefotaxime; and (d) oxidative stress under 0.5, 1, 1.5, or 2 mM H₂O₂. Data were presented as mean± SD from three replicates. Tukey’s post-test was used for statistical analysis, with * representing p < 0.05, ** representing p < 0.01, and *** representing p < 0.005 in one-way ANOVA.

Figure 3.

IscR-tmRNA regulatory axis mediates the sensitivity to oxidative stress. Overnight cultures with an initial OD₆₀₀ value of 0.6 were subjected to oxidative stress conditions with final concentrations of 0.5, 1.0, 1.5, or 2.0 mM H₂O₂ at 30°C with shaking for 30 min. The survivals were collected, washed with PBS (pH 7.4), and analysed by: (a) spotting 3 uL of each of ten-fold serial dilutions of the suspended bacteria onto LB agar plates supplemented with 50 μg/mL ampicillin and the growth of colonies on each plate was imaged after an incubation at 30°C for 16–20 h; and (b) spreading 100 μL of the 10⁻³-fold diluted bacteria suspension onto LB agar plates supplemented with 50 μg/mL ampicillin and enumerating the CFU after an incubation at 30°C for 16–20 h.

Figure 4.

IscR modulates the expression profile of tmRNA in response to oxidative stress. The expressions of iscR, ssrA and smpB genes under 0.5, 1.0, 1.5, or 2.0 mM H₂O₂ challenges were analysed by RT-qPCR in WT (A), ΔiscR (B), and ΔiscR/pBBR-iscR (C) strains. Error bars represented standard deviations of triplicate experiments. Tukey’s post-test was used to assess statistical significance with * representing p < 0.05 in one-way ANOVA.

Figure 5.

IscR-tmRNA regulatory axis influences motility and surface appendages formation in A. veronii. (a) Swimming and swarming motility assay of the wild type A. veronii and the derivative strains. 1 μL of the culture of 10⁸ cells/mL was spotted onto a 0.3% agar plate (for swimming assay) or a 0.5% agar plate (for swarming assay) and the images were captured after a incubation at 30°C for 6–8 h. (b) Morphological observation of the surface appendages of the wild type A. veronii and the derivative strains using the transmission electron microscopy (TEM). A 10 μL aliquot of the overnight culture was dropped onto the copper grid coated with carbon film, incubated with 2% phosphotungstic acid for 1–2 min, washed twice with ddH₂O, dried at room temperature, and observed using a HT7800 transmission electron microscope. The arrows indicated the flagella or fimbriae. Scale bars represented 500 nm.

Figure 6.

IscR-tmRNA regulatory axis influences the auto-aggregation, adhesion and cytotoxicity of A. veronii. (a) Bacterial aggregation was evaluated by observation of cell auto-aggregation formed after static overnight growth of the wild type A. veronii and the derivative strains at 30°C in liquid cultures. The OD₆₀₀ value of the supernatant was measured at 20 min intervals. (b) The adherence of A. veronii wild type and its derivative strains to in vitro Caco-2 intestinal epithelial cells (the left panel) and ex vivo murine intestinal tissues (the right panel) were plotted by the ratio of the survival colony counts to the initial number of infecting bacteria, or by the logarithmic function of the ratio of the survival colony counts to the weight of intestinal tissues, respectively. (c) Murine fibroblast L929 cells were incubated for 2-h at 37 °C with 5% CO₂ with A. veronii wild type and its derivative strains (the left panel), or their spent culture supernatants (the right panel), optionally treated with 50 ug/mL gentamicin to kill the bacteria, and the cytotoxic effects were plotted by the viability of the infected cells in CCk-8 assay. (d) Murine fibroblast L929 cells were incubated for 2-h at 37 °C with 5% CO2 with A. veronii wild type and its derivative strains, subsequently treated with 50 ug/mL gentamicin to kill the bacteria, and the morphology of L929 cells was observed under a phase-contrast microscope at 2-h post-infection. Error bars in (A), (B), and (C) represented the standard deviations of the means from experiments performed in triplicate. Tukey’s post-test was used, with * representing p < 0.05, ** representing p < 0.01, *** representing p < 0.005, and **** representing p < 0.001 in one-way ANOVA.

Figure 7.

IscR-tmRNA regulatory axis mediates the inter-bacteria competition capability. eGFP-labelled Escherichia coli DH5α and individual of A. veronii wild type or its derivative strains were co-incubated onto LB agar at a ratio of 1:1 for 6 h, and then the survival of the prey DH5α was analysed by: (a) spotting 3 uL of each of ten-fold serial dilutions of the suspended bacteria onto LB agar plates supplemented with 50 ug/mL gentamicin and the growth of the colony of the target DH5α on each plate was imaged after an incubation at 30° for 16–20 h; (b) spreading 100 μL of the 10⁻³-fold diluted bacteria suspension onto LB agar plates supplemented with 50 ug/mL gentamicin and enumerating the CFU of the target DH5α after an incubation at 30°C for 16–20 h; and (c) washing the bacteria with 1 mL PBS and detecting for the eGFP fluorescence density of the target DH5α using the fluorescent 1 (FL1) detector (525 nm band-pass filter). Error bars in (B) represented the standard deviations of the means from experiments performed in triplicate. Tukey’s post-test was used, with * representing p < 0.05 and ** representing p < 0.01 in one-way ANOVA.

Figure 8.

IscR-tmRNA regulatory axis is required for the full virulence of A. Veronii. KM mice were intraperitoneally inoculated with 2 × 10⁵ CFU/g of PBS-washed A. veronii wild type and its derivative strains. At 5 days post-inoculation, tissues from the heart, kidney, liver, lung, and spleen were collected, homogenized, and the CFU counts were determined. Each symbol represented one animal. The data presents the result of three independent experiments. Tukey’s post-test was used, with * representing p < 0.05 and ** representing p < 0.01 in one-way ANOVA. Dashes represented the geometric mean.

Figure 9.

IscR-tmRNA regulatory axis plays a key role in multiple stress response and pathogenicity in A. Veronii. Diagrammatic representation showing the positive regulation of IscR on the expression of tmRNA and the roles of IscR-tmRNA regulatory axis in multiple stress response and pathogenicity in A. Veronii.