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. 2019 May 16;26(5):745-755.e7.
doi: 10.1016/j.chembiol.2019.02.011. Epub 2019 Mar 21.

Multi-metal Restriction by Calprotectin Impacts De Novo Flavin Biosynthesis in Acinetobacter baumannii

Jiefei Wang  1 , Zachery R Lonergan  2 , Giovanni Gonzalez-Gutierrez  3 , Brittany L Nairn  2 , Christina N Maxwell  4 , Yixiang Zhang  5 , Claudia Andreini  6 , Jonathan A Karty  7 , Walter J Chazin  4 , Jonathan C Trinidad  8 , Eric P Skaar  9 , David P Giedroc  10
Affiliations

Multi-metal Restriction by Calprotectin Impacts De Novo Flavin Biosynthesis in Acinetobacter baumannii

Jiefei Wang et al. Cell Chem Biol. .

Abstract

Calprotectin (CP) inhibits bacterial viability through extracellular chelation of transition metals. However, how CP influences general metabolism remains largely unexplored. We show here that CP restricts bioavailable Zn and Fe to the pathogen Acinetobacter baumannii, inducing an extensive multi-metal perturbation of cellular physiology. Proteomics reveals severe metal starvation, and a strain lacking the candidate ZnII metallochaperone ZigA possesses altered cellular abundance of multiple essential Zn-dependent enzymes and enzymes in de novo flavin biosynthesis. The ΔzigA strain exhibits decreased cellular flavin levels during metal starvation. Flavin mononucleotide provides regulation of this biosynthesis pathway, via a 3,4-dihydroxy-2-butanone 4-phosphate synthase (RibB) fusion protein, RibBX, and authentic RibB. We propose that RibBX ensures flavin sufficiency under CP-induced Fe limitation, allowing flavodoxins to substitute for Fe-ferredoxins as cell reductants. These studies elucidate adaptation to nutritional immunity and define an intersection between metallostasis and cellular metabolism in A. baumannii.

Keywords: 3,4-dihydroxy-2-butanone 4-phosphate synthase (DHBPS); Acinetobacter baumannii; antimicrobial activity; calprotectin; host-microbe interaction; nutritional immunity; riboflavin biosynthesis; transition metal homeostasis.

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Conflict of interest statement

Declaration of Interests

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Transcriptomic analysis of WT and WT+CP A. baumannii.
(A) Metal content in the LB-Tris buffer growth media treated with 0 (grey) or 200 μg/mL CP (red). (B) RNAseq analysis of untreated WT vs. 200 μg/mL calprotectin treated WT (WT+CP) A. baumannii cells from 4 biological replicates. The fold-change in expression for each locus tag is indicated (see Table S1 for a complete list of genes). Gene names are indicated according to the NCBI annotation; otherwise, the locus tag (A1S_xxxx where ‘xxxx’ represents the locus tag number) is indicated. Genes regulated by the Zn uptake regulator (Zur) (yellow symbols), involved in Fe homeostasis (green symbols), involved in Cu uptake (red symbols) and associated with the histidine utilization (hut) operon (purple circles) (Nairn, et al., 2016) are highlighted. (C) qRT-PCR validation of selected genes identified by RNA-seq. A1S_0092, putative ferric siderophore uptake protein; A1S_0170, OprC outer membrane copper receptor; A1S_0242, FeoA; A1S_0652, FeoA; A1S_1214, benzoate 1,2-dioxygenase β subunit; A1S_1647, siderophore biosynthesis protein; A1S_1844, CatC3, muconolactone δ-isomerase; A1S_1868, porin for benzoate transport; A1S_2382, BasD; A1S_2580, siderophore biosynthesis protein. . *p < 0.05 as determined by Student’s t test with hypothetical value of 1. Data are the mean combined from 3 independent experiments ± S.D. See also Figure S1; Table S1.
Figure 2.
Figure 2.. LC-MS/MS proteomic analysis for WT and WT+CP A. baumannii.
Protein profiles of untreated WT vs. 200 μg/mL calprotectin treated WT (WT+CP) A. baumannii cells from 4 biological replicates. (A) Venn diagram for proteins detected at least 3 times in 4 replicates. (B) Histogram plot of the distribution of normalized abundance for all proteins detected in the untreated WT strain, as representative of all 4 growth conditions (see Figure S2A-D). The top-most 50% abundant proteins are indicated with red dash line with the bars shaded red. (C) Volcano plot for proteins detected at least 3 times for each condition among 4 biological replicates. The significance threshold was set at p≤0.001 and fold-change in protein abundance at >1.3 as shown in red dash box for proteins are up (right top) and down (left top) in WT+CP. Filled circles are shaded yellow for Zn-binding proteins, green for proteins involved in iron homeostasis, purple for Cu-binding proteins and pink for proteins involved in transcription or translation. (D) Proteins that are strongly changed in WT+CP conditions. Normalized abundance for proteins that was detected only in WT or WT+CP in all 4 replicates with the significance threshold set at the top-most 50% abundance (see Figure 2B). See also Figure S2; Table S2–3.
Figure 3.
Figure 3.. Calprotectin (CP) induces metal starvation in A. baumannii.
Growth yield at 4 h (OD 600 nm) (A) of cell cultures treated with 0 or 200 μg/mL CP when cell samples were collected for determination of total cell-associated metal by ICP-MS (panels B, C). Total cell-associated metal of WT and ΔzigA cells are shown in (B) for zinc (Zn) and iron (Fe) and (C) for other transition metals. The results shown reflect the mean of 3 independent replicates ± S.D. *p≤0.05 as determined by Student’s t test. (D) Rationale and design of this study using ΔzigA as a tool to study the effects of extreme metal limitation. See also Figure S3.
Figure 4.
Figure 4.. LC-MS/MS proteomic analysis for WT and ΔzigA A. baumannii.
Protein profiles of WT (WT+CP) vs. ΔzigA (ΔzigA+CP) A. baumannii cells treated with 200 μg/mL calprotectin from 4 biological replicates. (A) Venn diagram and (B) Volcano plot for proteins detected at least 3 times under each condition in 4 replicates. The significance threshold was set at p≤0.001 and fold-change in protein abundance at >1.3 as shown in red dash box. Yellow, Zn-binding proteins. (C) Proteins that are strongly changed in WT+CP and ΔzigA+CP cells. Normalized protein abundance for proteins that were only detected in WT+CP or ΔzigA+CP for in all 4 replicates with the significance threshold was set at the top-most 50% abundance (see Figure S2A-D). (D) Normalized protein abundances of enzymes of the flavin biosynthetic (rib) pathway (compare to Figure S4F) in the WT (grey) or WT+CP (red) cells. * p≤0.05 as determined by Student’s t test. Except for the RibF, which was only detected twice out of 4 replicates in WT, all proteins shown were detected in at least 3 of the 4 biological replicates. The mean of independent replicates ± S.D. is shown. (E) Riboflavin biosynthesis pathway in A. baumannii. Metabolites are indicated in bold and ZnII metalloenzymes are highlighted in blue. See also Figure S4; Table S4–5.
Figure 5.
Figure 5.. Biochemical characterization of RibBX.
(A) Michaelis-Menten plot for DHBPS activity with various substrate Ru5P concentrations. Error bars represent S.D. from 3 replicates. RibBX is in red. RibB (blank) is the authentic DHBPS in A. baumannii. (B) Michaelis-Menten plot for GCHII activity with various substrate GTP concentrations. Error bars represent S.D. from 3 replicates. RibBX is in red. RibA (blank) is the authentic GCHII in A. baumannii. (C) ITC titration of FMN to RibBX. The panel is shown as a representative fitting of 3 replicates. (D) Inhibition of DHBPS activity with 200 μM FMN. Reaction rate is normalized to the value with 0 μM FMN. * p≤0.05 using Student’s t-test. The mean of 3 independent replicates ± S.D. is shown. See also Figure S5.
Figure 6.
Figure 6.. Crystal structure of A. baumannii RibBX.
(A) Superposition of structure of RibBX (bright orange and pale yellow) and Vibrio cholerae RibB (Islam et al., 2015) (VcRibB) in the D-ribulose 5-phosphate-bound form (blue). Substrate binding residues are indicated for RibBX (bold). (B) Superposition of structure of RibBX (pale cyan and pale yellow) and Mycobacterium tuberculosis RibBA (Singh, et al., 2013) (MtRibBA; Rv1415) in the apo form (magenta). Zinc (Zn) binding residues in MtRibBA (stick representation). (C) Proposed dual regulatory model of the riboflavin biosynthesis pathway that becomes operative under extreme metal limitation mediated by CP. See also Figure S5; Table S6.
Figure 7.
Figure 7.. Riboflavin can rescue the growth of ΔzigA.
(A) Growth of ΔribBX and ΔzigA is impaired with 40 μM TPENThe mean of at least 3 independent replicates ± S.D. is shown. (B) Constitutive expression of ribBX in the presence of 20 μM TPEN improves growth of WT A. baumannii but impairs growth of ΔzigA. (C) Cellular FAD levels are notably decreased only in the ΔzigA strain in the presence of CP. (D) Riboflavin partially rescues the growth phenotype of ΔzigA with fumarate as sole carbon source in Zn-deplete conditions. See text for additional details. * p ≤ 0.05, * p ≤ 0.01 and **** p < 0.0001 as determined by one-way ANOVA with Tukey multiple comparisons test. The mean of at least 3 independent replicates ± S.D. is shown. See also Figure S5–7.
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