The Acinetobacter baumannii Znu System Overcomes Host-Imposed Nutrient Zinc Limitation

Abstract

Acinetobacter baumannii is an opportunistic bacterial pathogen capable of causing a variety of infections, including pneumonia, sepsis, wound, and burn infections. A. baumannii is an increasing threat to public health due to the prevalence of multidrug-resistant strains, leading the World Health Organization to declare A. baumannii a "Priority 1: Critical" pathogen, for which the development of novel antimicrobials is desperately needed. Zinc (Zn) is an essential nutrient that pathogenic bacteria, including A. baumannii, must acquire from their hosts in order to survive. Consequently, vertebrate hosts have defense mechanisms to sequester Zn from invading bacteria through a process known as nutritional immunity. Here, we describe a Znuptake (Znu) system that enables A. baumannii to overcome this host-imposed Zn limitation. The Znu system consists of an inner membrane ABC transporter and an outer membrane TonB-dependent receptor. Strains of A. baumannii lacking any individual Znu component are unable to grow in Zn-starved conditions, including in the presence of the host nutritional immunity protein calprotectin. The Znu system contributes to Zn-limited growth by aiding directly in the uptake of Zn into A. baumannii cells and is important for pathogenesis in murine models of A. baumannii infection. These results demonstrate that the Znu system allows A. baumannii to subvert host nutritional immunity and acquire Zn during infection.

Keywords: Acinetobacter; metal transporters; zinc.

FIG 1

The A. baumannii genome contains genes for an inner and outer membrane Zn acquisition system. (A) Schematic of the genomic context in A. baumannii ATCC 17978 of genes predicted to be involved in Zn acquisition and the putative functions of the encoded proteins. (B) Model with the predicted locations of each Znu system component. (C) ΔznuA pznuA-myc and ΔznuC pznuC-FLAG strains were grown in 30 μM TPEN, and localization of ZnuA-myc and ZnuC-FLAG was determined by immunoblot on cellular fractions. Blots shown are representative of at least three independent experiments, and a fractionation control is shown in Fig. S1 in the supplemental material.

FIG 2

The ZnuABC system is required for growth in low Zn conditions. WT, ΔznuA, ΔznuB, and ΔznuC strains were grown in LB (A), LB + 40 μM TPEN (B), and LB + 40 μM TPEN + 40 μM ZnCl₂ (C), and OD₆₀₀ was monitored over time. (D) WT, ΔznuA, ΔznuB, and ΔznuC strains harboring either an empty pWH1266 expression vector or pWH1266 providing a copy of the listed gene in trans were grown in LB + 40 μM TPEN and 75 μg/ml carbenicillin, and OD₆₀₀ was monitored over time. Curves are shown in biological triplicate as mean ± standard deviation (SD) and are representative of at least three independent experiments.

FIG 3

ZnuA is produced during, and required for, growth in low Zn conditions. (A) ΔznuA strains harboring pWH1266 alone or pWH1266 expressing znuA under the control of its native promoter were grown in either LB or LB + 30 μM TPEN, and 10 μg of cell lysate was added to each lane for immunoblotting. Equal loading of lanes is shown in Fig. S2 in the supplemental material, and the blot shown is representative of four independent experiments. (B) WT cells harboring pWH1266 and ΔznuA strains harboring an empty vector, a vector containing a WT copy of znuA, or a vector containing znuA with a substitution of A for H at residue 41 were grown in 40 μM TPEN and 75 μg/ml carbenicillin, and OD₆₀₀ was monitored over time. Expression of the ZnuA(H41A)-myc protein is demonstrated with an α-myc immunoblot in Fig. S2 in the supplemental material. Data are shown in biological triplicate ± SD and are representative of three independent experiments.

FIG 4

ZnuD is required for growth in low Zn conditions. WT and ΔznuD strains were grown in LB (A) and LB + 40 μM TPEN or LB + 40 μM TPEN + 40 μM ZnCl₂ (B), and OD₆₀₀ was monitored over time. (C) WT and ΔznuD strains harboring empty pWH1266 expression vector or pWH1266 containing znuD were grown in LB + 20 μM TPEN, and OD₆₀₀ was monitored over time. Data are shown in biological triplicate as mean ± SD and are representative of three independent experiments.

FIG 5

The Znu system contributes to growth during nutrient limitation imposed by the host protein calprotectin. WT, ΔznuA, ΔznuB, ΔznuC, and ΔznuD strains were grown for 6 h in 150 μg/ml recombinant human calprotectin, and OD₆₀₀ was measured to determine growth compared to untreated. ****, P < 0.0001 by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparison test. Data shown in biological triplicate as mean ± SD.

FIG 6

The Znu system is required for efficient Zn uptake. WT, ΔznuA, ΔznuB, ΔznuC, and ΔznuD strains were grown in 30 μM TPEN to mid-exponential phase when 5 μM ⁷⁰Zn was spiked into each culture and incubated 5 additional minutes. Cells were then pelleted, washed, and digested for quantification of metal levels by ICP-MS. ⁷⁰Zn uptake is normalized to sulfur content of each cell pellet. **, P < 0.01; ****, P < 0.0001 by one-way ANOVA followed by Dunnett’s multiple-comparison test. Each symbol represents an independent biological replicate, error bars ± SD.

FIG 7

ZnuA and ZnuD contribute differentially to A. baumannii survival in specific tissues during sepsis. Mice were infected retro-orbitally with the WT, ΔznuA, or ΔznuD strain and bacterial burdens in organs assessed at 24 h postinfection in the lungs (A), spleens (B), kidneys (C), livers (D), and hearts (E). Data shown as mean ± SD with each symbol representing bacterial burdens in the specified organ from an individual mouse. The y axis begins at the limit of detection in each organ, and gray symbols represent mice with bacterial burdens below the limit of detection. *, P < 0.05 by one-way ANOVA followed by Dunnett’s multiple comparison test; ****, P < 0.0001 by one-way ANOVA followed by Dunnett’s multiple comparison test.

FIG 8

ZnuA contributes to A. baumannii survival in the spleen during pneumonia. Mice were intranasally infected with a 1:1 mixture of either WT:ΔznuA strain (A, B) or WT:ΔznuD strain (C, D), and bacterial burdens were assessed at 36 h postinfection in the lungs (A, C) and the spleen (B, D). Data shown as mean ± SD with each symbol representing bacterial burdens in the specified organ from an individual mouse. The y axis begins at the limit of detection, and gray symbols represent mice with bacterial burdens below the limit of detection. *, P < 0.05 by unpaired t test.