Fig 1.
A. baumannii A1S_0934 is a COG0523 protein with conserved metal-binding and GTPase motifs.
(A) Phylogram ClustalX analysis of A1S_0934 (MigC) amino acid sequence conservation from 33 homologs identified in a previously published Sequence Similarity Network analysis[30]. MigC is represented by the UniProt ID A0A031LLB2. Multiple sequence alignment radiates outward from amino acids 1-200, indicating the amino acids from the GTPase/CobW domain. Gecos visualization by pairwise comparison of the (B) Walker A, (C) CxCC, and (D) Walker B domains of the SSN-identified homologs of A1S_0934 [30].
Fig 2.
A1S_0934 is a COG0523 Zn-binding GTPase.
(A) Competitive metal binding titration with A1S_0934 and mag-fura-2 (MF2) quantifies the second binding site of A1S_0934 for Zn2+ measuring absorbance in the UV-visible spectrum at 324 nm and 366 nm. Representative titration of Zn into 9.09 µM A1S_0934 and 9.48 µM MF2. Curve fitting (solid line) determined A1S_0934 KZn2 = 6.9 (± 0.2) × 106 M-1. Dashed lines represent simulations with 10-fold lower and 10-fold higher binding affinities to illustrate the robustness of curve fitting. (B-D) Representative competitive metal binding titrations with A1S_0934 and quin-2 quantify the high affinity binding site of A1S_0934 under various conditions by fluorescence intensity at 490 nm. Curve fitting (solid line) determined A1S_0934 without nucleotide to have KZn1 = 7.0 (± 0.5) × 1010 M-1, A1S_0934 in the presence of GDP to have Kzn1 = 1.5 (±0.1) × 1012 M-1, and A1S_0934 in the presence of GTP to have KZn1 = 2.7 (±0.4) × 1012 M-1. Titrations of A1S_0934 in the presence of GTP fit to a two-state binding model with Kzn2 = 5.7 (±1.3) × 109 M-1. Dashed lines represent simulated models with 10-fold lower and 10-fold higher KZn1 binding affinities to illustrate the robustness of curve fitting. Representative titrations are shown of Zn titrated into (B) 4.37 µM A1S_0934 and 8.69 µM quin-2 (C) 5.21 µM A1S_0934, 7.89 µM quin-2, and 1 mM GDP (D) 4.64 µM A1S_0934, 4.65 µM quin-2, and 1 mM GTP. (E) Normalized XANES (X-ray absorption near edge structure) for ZnII-bound A1S_0934 in NaCl (blue) and NaBr (red). (F-I) Zn EXAFS (Extended X-ray absorption line structure) and Fourier transformation of A1S_0934 in NaCl (F-G) and NaBr (H-I). (J) Malachite green assay measuring rate of NTP (nucleotide-triphosphate) hydrolysis of 5 µM A1S_0934 in the presence of Zn (5 µM) or Mn (500 µM) as indicated. (K) Michaelis-Menten kinetics of 5 µM Zn bound A1S_0934 for ATP and GTP. Curve fitting (solid line) was performed to get enzyme kinetic parameters. Reactions with GTP calculated Vmax = 0.59 ± 0.06 µmol/min/µmol A1S_0934 and Km = 110 ± 14 µM GTP. Reactions with ATP calculated Vmax = 0.175 ± 0.006 µmol/min/µmol A1S_0934 and Km = 51.1 ± 12 µM ATP. (L) Malachite green assay measuring rate of GTP hydrolysis of WT A1S_0934 and Walker B mutant E99A A1S_0934 in the presence and absence of stoichiometric Zn. **p<0.01, ****p<0.0001 by one-way ANOVA.
Fig 3.
A1S_0934 is required for full growth of A. baumannii during Zn-limitation.
(A) WT or Δ0934 integration controls (IC) or Δ0934::0934 were grown in LB ± 60 μM TPEN with OD600 monitored over time. (B) Percentage of rich medium (LB) growth as determined by OD600 at 15 h after growth in either 60 μM TPEN or 60 μM TPEN ± 0.5 μM ZnCl2 compared with untreated strains. ****p < 0.0001 by one-way ANOVA. (C) Percentage of rich medium (LB) growth as determined by OD600 at 15 h after growth in 60 μM TPEN of WT (IC), Δ0934 (IC), Δ0934::0934, Δ0934:: MB (C71A, C73A, and C74A), or Δ0934::E99A with OD600 monitored over time. (D) WT or Δ0934 containing an integration control (IC) or Δ0934::0934 were grown ± 250 µg/ml His6 knockout (SIIKO) calprotectin and OD600 was monitored over time. (E) Transmission Electron microscopy (TEM) of WT or Δ0934 cells in LB ± 40 µM TPEN. Cells were further assessed for (F) cell diameter and (G) peptidoglycan width using ImageJ software. *p < 0.05, **p < 0.01, ****p < 0.0001 by one-way ANOVA. (H) Confocal imaging of WT or Δ0934 grown in M9 minimal medium ± 10 μM TPEN with a HADA fluorescent amino acid probe. Cells were further assessed for (I) cell surface area and (J) mean fluorescence intensity using ImageJ software. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA.
Fig 4.
MigC interacts with and decreases the activity of MurD.
(A) Yeast two-hybrid assay assessing interacting proteins with MigC. Teal shading indicates the minimal interaction domain of MurD with MigC. (B) Michaelis-Menten kinetics of 40 nM MurD with varied ATP concentrations measuring UMAG production by HPLC. Reactions were performed with bovine serum albumin (BSA) added (black) and with 4 µM Zn MigC added (blue). Curve fitting of reactions with added BSA calculated Vmax = 432 ± 31 µmol/min/µmol MurD and Km = 149 ± 25 µM ATP. Curve fitting of reactions with added Zn MigC calculated Vmax = 232 ± 16 µmol/min/µmol MurD and Km = 144 ± 31 µM. (C) Inhibition curves measuring the relative activity of MurC and MurD with various Zn MigC concentrations. MurC and MurD activity were quantified by measuring MurC product, UMA, and MurD product, UMAG, by HPLC. Curve fitting of MurD data (solid line) determined the Ki for Zn MigC to be 32 ± 6 µM. A value for Ki could not be determined for MurC data. Dotted line represents the average relative activity of MurC measurements. (D) Malachite green assay measuring the rate of GTP hydrolysis of 5 µM Zn MigC in the absence and presence of MurD at the indicated concentrations (n = 3). Significance was assessed by one-way ANOVA.
Fig 5.
Disrupting MigC-MurD interactions alters A. baumannii morphological plasticity, antibiotic resistance, and colonization.
(A) WT murD KD or (B) ΔmigC murD KD were grown in LB with or without 25, 50, or 75 ng/mL anhydrotetracycline (AhTc) added. (C) WT murD KD and ΔmigC murD KD were grown in ± 75 ng/mL AhTc and then stained with a DAPI DNA dye and imaged on a confocal microscope. DIC images were captured simultaneously and overlaid. (D) These same strains were grown ± 100 ng/mL AhTc with OD600 monitored over time. (E) WT murD KD and ΔmigC murD KD were grown ± 75 ng/mL AhTc until mid-exponential phase and then were serial-diluted to plates containing 75 µg/mL carbenicillin to assess viability. ***p < 0.001, ****p < 0.0001 by one-way ANOVA. (F) These same strains were grown ± 50 ng/mL AhTc ± 20 μM TPEN with OD600 monitored over time. (G) WT and ΔmigC integration controls and the complementation strain were grown in LB ± either 15 μg/ml or 5 μg/ml of Ceftriaxone (CRO) and then plated to LBA and bacterial burdens were quantified. *p < 0.05 by one-way ANOVA. (H-I) Mice were intranasally infected with WT or ΔmigC. Bacterial burdens were assessed at 36 hpi in the (I) lungs or (J) heart. Data are represented as the mean ± SEM with each point indicating the bacterial burdens from an individual mouse in a specific organ. The limit of detection is indicated as LOD. ***p < 0.001, ****p < 0.0001 by unpaired t test.