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Zinc is a micronutrient required for numerous cellular functions in all living organisms. In recent years, zinc as an alternative additive is increasingly used in farmed animals to enhance growth performance, as it is able to prevent or reduce the duration and severity of diarrhea caused by some pathogens, especially pathogenic E. coli. It has been reported that zinc supplementation also leads to an increased rate of antibiotic resistance in commensal E. coli. However, until now the underlying mechanisms of these effects driven by zinc exposure have remained unclear. In our studies, we characterized two E. coli strains, commensal strain IMT29408 and pathogenic strain IMT8073 (aEPEC) defined by genotypic analysis, as representatives of porcine intestinal E. coli to investigate zinc sensitivity, the effects of zinc on growth rates, intracellular zinc content of commensal strain in vitro and protein expression differences induced by zinc. Since there is only limited available knowledge about laboratory strains (K-12) instead of wild-type strains that are highly different in gene content and consequent physiological properties compared to lab strains.
In the present study, the bacteria were cultured in LB medium using a bioreactor under anaerobic condition with or without 1 mM zinc chloride determined on the basis of minimal inhibitory concentration test. There were no significantly inhibitory influences of zinc on cell viability represented by CFU per milliliter and cell turbidity represented by OD600 values from 1 h to 7 h in commensal strain IMT29408. By contrast, although the cell viability of aEPEC IMT8073 was not significantly decreased by zinc, the bacteria turbidities were reduced by zinc treatment from 1 h to 7 h. These indicated that the inhibition of aEPEC virulence was likely not induced by the reduction in bacterial viability, but rather by changes in cell physiology.
In the commensal strain IMT29408, we investigated how the intracellular zinc content changes in the presence or absence of 1 mM zinc over time. The results showed that from 0 h to 10 h of incubation, the intracellular zinc content was about 8.6 x 10(6) atoms/cell in average with a maximum of 1.2 x 10(7) atoms/cell at 2 h in the presence of zinc, while cultured in the control (LB medium), the average value was about 1.1 x 10(5) atoms/cell with a peak amount of 4.5 x 10(5) atoms/cell at 3 h. The results suggested that the intracellular zinc content in commensal E. coli varies significantly with the changes in environmental zinc availability and exposure time.
Based on the growth rates and intracellular zinc content curves over time, bacterial samples were collected at 2 h and 5 h time points for proteomic analysis to investigate the differences of protein expression between zinc treatment and control. In commensal E. coli IMT29408, proteomic analysis using 2D-DIGE revealed 544 protein spots in total. We identified two differentially expressed (>1.5-fold change, p<0.05 (student’s t test)) proteins up-regulated (DegP and FabF) and seven proteins down-regulated (ManX, Mdh, Pgk, RbsB, RpoA, Tsf and YhdH) at 2 h of incubation with zinc using MALDI-TOF MS/MS. At 5 h of incubation, there were seven up-regulated (DegP, FabF, RplD, TnaA, TpiA, Tsx and YchF) and three down-regulated (AhpC, RffG and PflB) proteins identified. Most of them have not previously been implicated in responding to zinc compared with the available data about E. coli K-12, which are mainly involved in translation, stress defense, glycolysis and transport. We also identified the differentially expressed proteins only influenced by continuous zinc treatment (from 2 h to 5 h), six up-regulated and fourteen down-regulated. Among these, two porins OmpC and OmpX were down-regulated, which suggested that persistent zinc treatment could result in reduced outer membrane permeability. These data give evidence that reduced outer membrane permeability is a possible explanation for the zinc-induced antibiotic resistance.
In pathogenic E. coli IMT8073, there were 413 protein spots detected by 2D-DIGE. Out of them, at 2 h of incubation we identified two up-regulated proteins (KatE and ClpB), while at 5 h, there were eleven up-regulated (AccC, AsnS, AspA, AtpA, AtpD, DegP, FabF, HybC, PyrG, TnaA and Tsf) and six down-regulated (CadA, MinD, NanA, NusA, PflB and Udp) proteins identified. From these identified proteins, we concluded that pathogenic E. coli adapted to zinc stress by up-regulation of proteins associated with oxidative stress defense, translation, and energy generation (ATP synthesis, glycolysis, and anaerobic respiration) as a survival strategy. We also identified eight up-regulated and twenty-six down-regulated proteins only influenced by zinc when comparing 2 h to 5 h of incubation. Most of these proteins were involved in translation (PrfB and LysS up-regulated while Tuf, GlyS, TypA, RplI and GlnS down-regulated), metabolic processes (GlpQ, GlnA and AspA up-regulated while FbaA, Pta, TdcE, PflB, MinD, Gnd, CadA, PspE and EutD down-regulated) and biosynthetic processes (PdxJ up-regulated while FabF, GlmS and HemX down-regulated). Of these proteins, TypA, a regulatory protein was down-regulated and this suggested that persistent zinc treatment probably influenced EPEC virulence, as typA-mutants of EPEC
showed declined adherence to cultured cells. Based on these results, it was indicated that the antibacterial effects of zinc on EPEC are likely achieved by a combination of multiple physiological mechanisms such as oxidative stress, envelope stress and metabolic influences, which might be a possible explanation for previous findings that zinc treatment reduces virulence of EPEC.
Comparatively, the differentially expressed proteins influenced by zinc were mostly different among the wild-type E. coli strains (commensal and pathogenic) in the present study as well as the E. coli K-12 described previously. This indicated that the metabolic strategies for adaptation of zinc stress vary among these strains, which is probably due to the different number of protein-coding genes involved in physiological activities.