Zinc uptake system ZnuACB is essential for maintaining pathogenic phenotype of F4ac+ enterotoxigenic E. coli (ETEC) under a zinc restricted environment

Abstract

Zinc is the second trace element of living organisms after iron. Given its crucial importance, mammalian hosts restrict the bioavailability of Zinc ions (Zn²⁺) to bacterial pathogens. As a countermeasure, pathogens utilize high affinity Zn²⁺ transporters, such as ZnuACB to compete with the host for zinc. It is essential for bacteria to maintain zinc homeostasis and thus maintain their physiology and pathogenesis. In an attempt to uncover the zinc transporter in F4⁺ enterotoxigenic E. coli (ETEC) C83902, we analyzed two RNA-seq data sets of bacteria samples when different zinc treatments (restriction or abundance) were applied. Considering data revealing that the high affinity zinc uptake system ZnuACB acts as the main transporter in ETEC C83902 to resist zinc deficiency, we deleted znuACB genes to study the role of them in ETEC C83902. The deletion of znuACB genes results in growth perturbation and a sharp decrease in the ability of biofilm formation and adhesion of bacteria in vitro. Taking the data together, this study demonstrates that the ZnuACB system is required for ETEC C83902 to acquire zinc, which highly contributes to ETEC pathogenicity as well.

Keywords: Enterotoxigenic E. coli (ETEC); Zinc deficiency; ZnuACB; pathogenicity.

Figure 1

Validation of expression profiling data by RT-qPCR. A The diagram of the operon encoding ZnuACB transporter. B DEGs were validated by RT-qPCR. The relative gene expression level was calculated using the 2^−ΔΔCt value method and normalized by the gapA housekeeping gene. * significant at p < 0.05, ** significant at p < 0.01 and *** significant at p < 0.001. Data presented as mean ± standard deviations of three independent experiments. C Comparison of 7 DEG between RNA-Seq and RT-qPCR data. The y axes represent the log2 (fold change) measured by RNA-Seq and RT-qPCR, respectively.

Figure 2

Bacterial growth curves. A WT strain and mutants were incubated in LB broth at 37 °C for 10 h with agitation, and OD₆₀₀ optical was measured every 1 h. B WT strain and mutants were incubated in LB broth with 30 μM TPEN at 37 °C for 12 h with agitation. C and D WT strain and mutants were incubated in 30 μM TPEN pre-treated LB broth with 0.5 mM and 2 mM Zn²⁺ at 37 °C for 10 h with agitation, and OD₆₀₀ optical was measured every hour. Data are the means and standard deviations from three independent experiments. *p < 0.05 (Student t test) compared to the corresponding wild type.

Figure 3

Biofilm formation of WT strain and isogenic mutants of znuACB. A Quantification of biofilm formation of WT C83902 and C83902 ΔznuACB. Surface-adhered biofilm on 96 well microtiter plates was quantified by measuring OD₆₀₀ of ethanol-solubilized CV (2%) after biofilm staining. Data are shown as mean ± standard deviation of triplicate experiments. Significant differences between the mutant and WT C83902 are indicated by p < 0.001 ***. B The decrease of biofilm formation under zinc restriction was characterized by SEM. WT strain and C83902 ΔznuACB were fixed with 2.5% glutaraldehyde after incubation in BIM with or without 30 μM TPEN for 72 h. In order to eliminate the influence of DMSO, the TPEN solvent, BIM with DMSO was set as a control. Fixed bacteria were dehydrated by alcohol gradient, then dried by carbon dioxide critical point dryer and sprayed with gold, finally the sample biofilms were characterized by SEM. The biofilms are marked with a red arrow.

Figure 4

Adherence of WT C83902 and C83902 ΔznuACB. A Deletion of C83902 znuACB decreases the adherence to IPEC-J2 cell. An adherence assay of WT C83902 and C83902 ΔznuACB to IPEC-J2 cell after pre-incubation in LB medium. B Zinc restriction led to a significant decrease of adhesion ability of C83902 ΔznuACB, while zinc supplementation can restore its adhesion ability. Bacteria were incubated in 30 μM TPEN pre-treated LB medium and supplemented with 10 μM or 20 μM Zn²⁺, then adherence assays of WT C83902 and C83902 ΔznuACB to IPEC-J2 cell were performed. The WT strain adhesion index was assumed to be 100%. Data are expressed as mean ± standard deviation of triplicate experiments. Statistically significant differences are indicated as ***p < 0.001 when compared to the WT strain.