Prevention of gastrointestinal lead poisoning using recombinant Lactococcus lactis expressing human metallothionein-I fusion protein

Abstract

Low-level lead poisoning is an insidious disease that affects millions of children worldwide, leading to biochemical and neurological dysfunctions. Blocking lead uptake via the gastrointestinal tract is an important prevention strategy. With this in mind, we constructed the recombinant Lactococcus lactis strain pGSMT/MG1363, which constitutively expressed the fusion protein glutathione S-transferase (GST)-small molecule ubiquitin-like modifier protein (SUMO)-metallothionein-I (GST-SUMO-MT). The thermodynamic data indicated that the average number of lead bound to a GST-SUMO-MT molecule was 3.655 and this binding reaction was a spontaneous, exothermic and entropy-increasing process. The total lead-binding capacity of pGSMT/MG1363 was 4.11 ± 0.15 mg/g dry mass. Oral administration of pGSMT/MG1363 (1 × 10(10) Colony-Forming Units) to pubertal male rats that were also treated with 5 mg/kg of lead acetate daily significantly inhibited the increase of blood lead levels, the impairment of hepatic function and the decrease of testosterone concentration in the serum, which were all impaired in rats treated by lead acetate alone. Moreover, the administration of pGSMT/MG1363 for 6 weeks did not affect the serum concentration of calcium, magnesium, potassium or sodium ions. This study provides a convenient and economical biomaterial for preventing lead poisoning via the digestive tract.

Figure 1. The isothermal titration calorimetry (ITC) analyses of the reaction of recombinant proteins binding lead ions.

(A) ITC analyses of the reaction of GST-SUMO binding lead. (a) Represents the ITC raw data for 20 automatic injection of lead acetate solution (0.01 M) into the sample cell containing GST-SUMO solution (5×10⁻⁵ M). (b) Represents the plot and trendline of cumulative heat of injectant (corresponding to raw data of a) vs molar ratio of lead to GST-SUMO. (B) ITC analyses of the reaction of GST-SUMO-MT binding lead. (a) Represents the ITC raw data for 20 automatic injection of lead acetate solution (0.01 M) into the sample cell containing GST-SUMO-MT solution (5×10⁻⁵ M). (b) Represents the plot and trendline of cumulative heat of injectant (corresponding to raw data of a) vs molar ratio of lead to GST-SUMO-MT.

Figure 2. Lead-binding capacity analyses of MG1363, pGS/MG1363 and pGSMT/MG1363.

The L. lactis strains were grown in GM17 medium with or without erythromycin until the OD₆₀₀ reached 0.4. Then, lead acetate was added to a final concentration of 0.1 mM and the culture was incubated at 30 °C for up to 20 h. The cells were harvested and dried to constant weight. The mass of lead in these dried pellets was evaluated using ICP-OES (n = 3). The parameters are expressed as the means ± S.E.s. The curves represent the lead-accumulating tendency of the recombinant strains at different time points. The capital letter “R” represents the strains grown in the presence of erythromycin.

Figure 3. Growth tendency of L. lactis strains in the lead-containing medium.

The L. lactis strains were inoculated into GM17 medium containing 0.1 mM lead for 20 h. The wet weight mass and optical density (OD₆₀₀) of samples was measured at different time points (n = 4). The parameters are expressed as the means ± S.E.s. The asterisk indicates a significant difference at p < 0.05.

Figure 4. Morphological observations of recombinant L. lactis strains cultured with lead.

The MG1363, pGS/MG1363 and pGSMT/MG1363 strains were cultured using GM17 medium containing a final concentration of 0.1 mM lead acetate at 30 °C. After a 20-h culture, the cells were collected and washed three times. The color observations of pellets at the end of culture ((A) side view; (B) top view); (C). The comparison diagram of average density scanned by Image J software. The parameters are expressed as the means ± S.E.s. (n = 3).

Figure 5. Fermentation of pGSMT/MG1363.

The fermentation of pGSMT/MG1363 was performed in a 10-L fermenter under optimal conditions (30 °C, pH 6.0, 100 rpm agitation and 1.0 vvm aeration). Changes in the parameters including optical density and lead-binding capacity of pGSMT/MG1363 during the process of a 10-L fermentation were recorded by time course. The parameters are expressed as the means ± S.E.s. (n = 4).

Figure 6

Preventative effects of pGSMT/MG1363 on hepatic (A–C) and renal (D–F) functions. The serum parameters are expressed as the means ± S.E.s. Control: rats without treatment; model: 5 mg/kg/d lead-treated rats; pGS/MG1363: lead-treated rats administered 1 × 10¹⁰ CFU/d; and pGSMT/MG1363-L, pGSMT/MG1363-M, and pGSMT/MG1363-H: lead-treated rats administered 1 × 10⁸, 1 × 10⁹, and 1 × 10¹⁰ CFU/d, respectively. n = 9. The asterisks, *, ** and ***, indicate significant differences at p < 0.05, p < 0.01 and p < 0.001, respectively.

Figure 7. Creatine kinase and testosterone concentrations of rats treated with lead and recombinant L. lactis strains.

The concentrations of creatine kinase and testosterone are expressed as the means ± S.E.s. Control: rats without treatment; model: 5 mg/kg/d lead-treated rats; pGS/MG1363: lead-treated rats administered 1 × 10¹⁰ CFU/d; and pGSMT/MG1363-L, pGSMT/MG1363-M, and pGSMT/MG1363-H: lead-treated rats administered 1 × 10⁸, 1 × 10⁹, and 1 × 10¹⁰CFU/d, respectively. n = 9. The asterisks, ** and ***, indicate significant differences at p < 0.01 and p < 0.001, respectively.

Figure 8. mRNA analysis of MT-I in the intestine after treatment with lead and recombinant L. lactis strains.

The relative expression levels of mt-I detected by RT-PCR were compared with that of rps 16 and expressed as the mean ± S.E.s. (n = 5).