An Acinetobacter baumannii, Zinc-Regulated Peptidase Maintains Cell Wall Integrity during Immune-Mediated Nutrient Sequestration

Abstract

Acinetobacter baumannii is an important nosocomial pathogen capable of causing wound infections, pneumonia, and bacteremia. During infection, A. baumannii must acquire Zn to survive and colonize the host. Vertebrates have evolved mechanisms to sequester Zn from invading pathogens by a process termed nutritional immunity. One of the most upregulated genes during Zn starvation encodes a putative cell wall-modifying enzyme which we named ZrlA. We found that inactivation of zrlA diminished growth of A. baumannii during Zn starvation. Additionally, this mutant strain displays increased cell envelope permeability, decreased membrane barrier function, and aberrant peptidoglycan muropeptide abundances. This altered envelope increases antibiotic efficacy both in vitro and in an animal model of A. baumannii pneumonia. These results establish ZrlA as a crucial link between nutrient metal uptake and cell envelope homeostasis during A. baumannii pathogenesis, which could be targeted for therapeutic development.

Keywords: Acinetobacter; antibiotics; calprotectin; infection; metals; nutritional immunity; peptidase; peptidoglycan; zinc.

Figure 1.. ZrlA Is a Peptidase Induced during Zn Starvation

(A) ZrlA expression was assessed in wild-type (WT) and ΔzrlA (15 μg protein/lane) by immunoblot after growth in lysogeny broth (LB) ± 40 μM TPEN. (B) Transcriptional changes for zrlA and other predicted d,d-CPases A1S_1248, A1S_2435, and A1S_2479 were assessed by qRT-PCR + 250 μg/ml CP. **p < 0.01 as determined by one-way ANOVA with Tukey multiple comparisons test on three independent experiments, means ± SD. (C) Modified cadmium-ninhydrin assay was performed on recombinant MBP-ZrlA in the presence of a peptide substrate. *p < 0.05, ***p < 0.001 as determined by one-way ANOVA with Tukey multiple comparisons test on three independent experiments, means ± SD. (D) ZrlA protein localization was assessed in WT and ΔzrlA (7.5 μg protein/lane) by immunoblot of membrane fractions following growth in LB ± 40 μM TPEN. (E) Co^II titration, where a tetrahedral or distorted tetrahedral coordination geometry is suggested with a d-d transition of 400 M⁻¹ • cm⁻¹ at 600 nm (Corwin and Koch, 1987). Inset, number of Co^II:ZrlA^42C mol • equivalents corresponding to each spectrum shown; note that all spectra acquired at Co^II:ZrlA^42C overlap, revealing a 1:1 metal binding stoichiometry. (F) Normalized binding titration of a mixture of ZrlA^42C and competitor quin-2 with Zn in two independent experiments: 17.0 μM ZrlA^42C with 15.2 μM quin-2 (blue filled circles) and 15.1 μM ZrlA^42C with 12.3 μM quin-2 (black filled circles). The continuous lines represent the results of a nonlinear, least-squares fit to a Zn:ZrlA^42C = 1:1 binding model. The global fitting results in K_Zn = 3.6 (±0.4) × 10¹¹ M^{− 1}. See also Figure S1.

Figure 2.. ZrlA Contributes to Cellular Morphology and Envelope Integrity

(A) Mean fluorescence of WT or ΔzrlA ± 10 μΜ TPEN after DA-EDA labeling of the fifth position of PG pentapeptides. ****p < 0.0001 as determined by one-way ANOVA with Tukey multiple comparisons test (n = 100 cells/group). (B) Mean fluorescence of WT or ΔzrlA ± 10 μM TPEN after HADA labeling of the fourth position of PG pentapeptides **p < 0.01, ****p < 0.0001 as determined by one-way ANOVA with Tukey multiple comparisons test (n = 100 cells/group). (C) Normalized abundance of PG muropeptide tripeptide monomers (sum M3, M3(−N-acetylglucosamine [GlcNAc]; see Figure S2 for a representative liquid chromatography-tandem mass spectrometry (LC-MS/MS) chromatogram and Table S1 for muropeptide designations and expected and experimental masses) in WT or ΔzrlA ± 40 μM TPEN. (D) Normalized abundance of muropeptide tetrapeptide monomers (sum M4,M4[–GlcNAc]) (Figure S2; Table S1) in WT or ΔzrlA ± 40 μM TPEN. *p < 0.05, **p < 0.01, ***p < 0.001 as determined by one-way ANOVA with Tukey multiple comparisons test on biological duplicates, means ± SD. (E) Microscopy of WT or ΔzrlA A. baumannii grown to mid-log in LB + 40 μM TPEN (100×). (F) Microscopy of WT or ΔzrlA A. baumannii grown to mid-log in LB (100×). Scale bar, 5 μm. (G) Ethidium bromide uptake following growth in LB. ***p < 0.001 as determined by Student’s t test of mean line slopes from three independent experiments. (H) Ethidium bromide uptake following growth in 40 μM TPEN, ****p < 0.0001 as determined by Student’s t test of mean line slopes from three independent experiments. (I) Percentage of growth as determined by optical density 600 (OD600) at 8 h of growth in LB, 0.01% SDS, or 0.01 mM EDTA for WT and ΔzrlA compared with untreated samples. ****p < 0.05 as determined by Student’s t test from three independent experiments, means ± SD. (J) ΔzrlA was subjected to qRT-PCR analysis of putative d,d-CPases in 250 μg/ml CP. *p < 0.05, ***p < 0.001 as determined by Student’s t test from three independent experiments, means ± SD. (K) ΔzrlA was subjected to qRT-PCR analysis of putative d,d-CPases in LB compared with expression in the WT strain. *p < 0.05 as determined by Student’s t test from three independent experiments, means ± SD. See also Table S1 and Figure S2.

Figure 3.. ZrlA Is Critical for Full Growth in Low Zn Environments and During Infection

(A) WT or ΔzrlA were grown ± 250 μg/ml CP with OD₆₀₀ monitored over time. (B) Percentage of growth as determined by OD₆₀₀ at 8 h after growth in 250 μg/ml CP ± 50 μM ZnCl₂ compared with untreated strains. *p < 0.05 as determined by Student’s t test from three independent experiments, means ± SD. (C) qRT-PCR analysis was performed on WT or ΔzrlA after growth in 250 μg/ml CP. **p < 0.01, ***p < 0.001 as determined by Student’s t test from three independent experiments, means ± SD. (D) Cellular ⁷⁰Zn was quantified by ICP-MS and normalized to ³⁴S for WT and ΔzrlA. *p < 0.05, ***p < 0.001 as determined by one-way ANOVA with Tukey multiple comparisons test from three independent experiments, means ± SD. (E) Percentage of growth as determined by OD₆₀₀ at 6 h for WT, Δzur, or ΔzrlA grown in 0.01% SDS/0.1 mM EDTA relative to untreated strains. **p < 0.01, ****p < 0.0001 as determined by one-way ANOVA with Tukey multiple comparisons test from three independent experiments, means ± SD. (F) Mice were intranasally infected with a 1:1 mixture of WT and ΔzrlA, and bacterial burdens were assessed at 36 hpi in the lungs. ****p < 0.0001 as determined by Mann-Whitney U test. (G) WT and ΔzrlA bacterial burdens recovered from the liver at 36 hpi. **p < 0.01 as determined by Mann-Whitney U test; error bars are median ± interquartile range (n = 16). See also Figure S3

Figure 4.. ZrlA Is Required for Overcoming Antibiotic Exposure In Vitro and In Vivo

(A-D) WT and Δzr/A were grown (A) ± carbenicillin, (B) ± vancomycin, (C) ± polymyxin B, and (D) ± tetracycline with OD₆₀₀ monitored overtime. (E) WT and Δzr/A were grown in the presence of carbenicillin ± TPEN with OD₆₀₀ monitored over time; data depict growth at 8 h relative to growth with no carbenicillin. ****p < 0.0001 as determined by one-way ANOVA with Tukey multiple comparison’s test from three independent experiments, means ± SD. (F) WT and Δzr/A were grown in the presence of polymyxin B ± TPEN with OD₆₀₀ monitored over time; data depict growth at 24 h relative to growth with no polymyxin B. ****p < 0.0001 as determined by one-way ANOVA with Tukey multiple comparisons test from three independent experiments, means ± SD. (G) WT or Δzr/A bacterial burdens recovered from the lungs at 36 hpi. ***p < 0.001, ****p < 0.0001 as determined by Mann-Whitney U test; error bars are median ± interquartile range (n = 20). (H) Competitive index ([input Δzr/A/WT])/[output Δzr/A/WT]) in the lungs at 36 hpi. ****p < 0.0001 as determined by Student’s t test with an arbitrary value of 1 or between treatments; error bars are median ± interquartile range (n = 20). See also Figure S4.