The tRNA methyltransferase TrmB is critical for Acinetobacter baumannii stress responses and pulmonary infection

Abstract

As deficiencies in tRNA modifications have been linked to human diseases such as cancer and diabetes, much research has focused on the modifications' impacts on translational regulation in eukaryotes. However, the significance of tRNA modifications in bacterial physiology remains largely unexplored. In this paper, we demonstrate that the m⁷G tRNA methyltransferase TrmB is crucial for a top-priority pathogen, Acinetobacter baumannii, to respond to stressors encountered during infection, including oxidative stress, low pH, and iron deprivation. We show that loss of TrmB dramatically attenuates a murine pulmonary infection. Given the current efforts to use another tRNA methyltransferase, TrmD, as an antimicrobial therapeutic target, we propose that TrmB, and other tRNA methyltransferases, may also be viable options for drug development to combat multidrug-resistant A. baumannii.

Keywords: Acinetobacter; iron acquisition; macrophages; oxidative stress; pneumonia; tRNA modification.

Fig 1

Loss of trmB does not significantly impact antibiotic susceptibility, but renders ARC6851 more susceptible to oxidative stress (A) Antibiotic susceptibility of trm mutants. Eight ARC6851 trm deletion mutants were screened for changes in MICs to a variety of antibiotics using a 2-fold broth dilution method. Mid-exponential cultures were normalized at OD₆₀₀ 0.01 and grown for 16 h with shaking at 37°C. MIC was determined as <10% growth compared to a non-treated culture. Greater than 2-fold changes were considered significant. (B) Oxidative stress resistance of trm mutants. Strains were grown to mid-exponential phase before being treated with 0 mM or 5 mM H₂O₂ for 2 h. Survival was measured by serial dilution and quantification of the recoverable CFU/mL. Fold changes were calculated against the wild-type strain.

Fig 2

TrmB is essential for resistance to oxidative and acid stresses. (A) H₂O₂ killing of ARC6851 strains. Wild-type, ΔtrmB, and trmB+ strains were grown to mid-exponential phase before being treated with 0 mM or 5 mM H₂O₂ for 2 h. Survival was measured by serial dilution and quantification of recoverable CFU/mL. Points represent technical replicates from at least three biological replicates, with a horizontal line representing the mean, and errors bars representing the standard error of the mean (SEM). *P < 0.05; one-way ANOVA, Tukey’s test for multiple comparisons. (B and C) Representative growth curves of wild type, ΔtrmB, and trmB+ in non-buffered LB at pH 5.0 and pH 7.0. Mid-exponential cultures were normalized to OD₆₀₀ = 0.01 and grown for 16 h with shaking at 37°C. ****P < 0.0001, unpaired t tests at 16 h for ΔtrmB compared to wild type and trmB+.

Fig 3

TrmB plays a significant role in replication within macrophages (A) J774A.1 macrophages were infected with mid-exponential phase ARC6851 wild-type, ΔtrmB, and trmB+ strains. Intracellular CFU were determined at 2, 4, and 6 h post-infection. *P < 0.05, ****P < 0.0001, mixed effects model, Tukey’s test for multiple comparisons. (B) Infected macrophages were fixed at 4 h post-infection. Number of bacteria per ACV were determined with at least 14 representative confocal microscopy images and two biological replicates per strain. (C) The samples were stained to detect cell nuclei (blue), A. baumannii (green), and actin (red). Bars 20 µM. Insets (13 µM) are a higher magnification of the area denoted in the white box of the corresponding image. Representative ACVs are indicated with pink (>six bacteria/ACV) and yellow (one to two bacteria/ACV) arrows.

Fig 4

ARC6851 ΔtrmB is attenuated in an acute murine pneumonia model. C57BL/6 mice were infected with ~5 × 10⁷ CFU of mid-exponential ARC6851 wild-type, ΔtrmB, and trmB+ strains. At 24 h post-infection, the lungs, kidneys, and spleens were harvested, and the bacterial load present in each tissue was determined with serial dilutions. Each symbol represents an individual mouse, and the horizontal bar represents the median. Data collected from three independent experiments. ****P < 0.0001, Kruskal-Wallis test.

Fig 5

A. baumannii TrmB performs the m⁷G tRNA modification. (A) m⁷G levels in ARC6851 wild-type, ΔtrmB, and trmB+ strains were determined with LC-MS. ΔtrmB m⁷G levels were below detection limit (ND). ****P < 0.0001, one-way ANOVA, Tukey’s test for multiple comparisons. (B) m⁷G levels in Ab04 wild-type, ΔtrmB, and trmB+ strains were determined with thin-layer chromatography and (C) analyzed with ImageQuant. *P < 0.05, one-way ANOVA, Tukey’s test for multiple comparisons.

Fig 6

TrmB plays a significant regulatory role in ARC6851 under oxidative stress. (A) Principal component analysis of ARC6851 wild-type, ΔtrmB, and trmB+ proteomes with and without H₂O₂ treatment. Mid-exponential cultures were treated with 0 mM or 5 mM H₂O₂ for 2 h before being pelleted, lysed, and acetone precipitated. Digested proteome samples were analyzed with reverse phase liquid chromatography-mass spectrometry. (B) (left) Volcano plot of Table 2 data showing differentially expressed proteins in ARC6851 wild type in H₂O₂ treatment versus non-treated. (right) Volcano plot of Table 3 data showing differentially expressed proteins in ARC6851 ΔtrmB in H₂O₂ treatment versus non-treated. Red dots are more than 2.5-fold upregulated, and blue dots are more than 2.5-fold downregulated. Large dots represent hits used for further analysis.

Fig 7

ARC6851 ΔtrmB fails to post-transcriptionally upregulate acinetobactin and is more susceptible to iron deprivation under H₂O₂ stress. (A) Relative gene expression of basB, basE, bauA, bauB, and OB946_15780 (OHRP) genes in ARC6851 wild-type (W), ΔtrmB (Δ), and trmB+ (+) strains grown in 2 mM H₂O₂ for 10 min (top) or 2 h (bottom) compared to 0 mM, as determined by qRT-PCR. Dotted line represents 2-fold change. (B) H₂O₂ killing of ARC6851 wild-type, ΔtrmB, and trmB+ strains in grown in LB, Tris minimal succinate (TMS) media, or Chelex-100-treated TMS (cTMS) with treatment in 3 mM H₂O₂ for 2 h compared to 0 mM. Survival was measured by serial dilution and quantification of recoverable CFU/mL. Points represent technical replicates from at least four biological replicates, with a horizontal line representing the mean, and errors bars representing the standard error of the mean (SEM). **P < 0.01; one-way ANOVA, Tukey’s test for multiple comparisons.