Identification and evaluation of a panel of strong constitutive promoters in Listeria monocytogenes for improving the expression of foreign antigens

Abstract

Attenuated Listeria monocytogenes could be a potential vaccine vector for the immunotherapy of tumors or pathogens. However, the lack of reliable promoters has limited its ability to express foreign antigens. In the present study, 21 promoters were identified from Listeria monocytogenes through RNA-seq analysis under two pH conditions of pH 7.4 and pH 5.5. Based on the constructed fluorescence report system, 7 constitutive promoters exhibited higher strength than P_help (1.8-fold to 5.4-fold), a previously reported strong promoter. Furthermore, the selected 5 constitutive promoters exhibited higher UreB production activity than P_help (1.1-fold to 8.3-fold). Notably, a well-characterized constitutive promoter P₁₈ was found with the highest activity of fluorescence intensity and UreB production. In summary, the study provides a panel of strong constitutive promoters for Listeria monocytogenes and offers a theoretical basis for mining constitutive promoters in other organisms. KEY POINTS: • Twenty-one promoters were identified from L. monocytogenes through RNA-seq. • Fluorescent tracer of L. monocytogenes (P₁₈) was performed in vitro and in vivo. • A well-characterized constitutive promoter P₁₈ could improve the expression level of a foreign antigen UreB in L. monocytogenes.

Keywords: Acid stress; Constitutive promoter; L. monocytogenes; RNA-seq.

Fig. 1

The screening of acid stress treatments for L. monocytogenes RNA-seq analysis. a The growth characteristics of L. monocytogenes at different pH conditions. The initial inoculation amount of L. monocytogenes was 3.6×10⁸ CFU/mL. b Acid resistance determination of L. monocytogenes with different acid stress treatments. The viable bacteria number under eight kinds of different acid stress (pH 4.5, 30 min; pH 4.5, 1 h; pH 5.0, 30 min; pH 5.0, 1 h; pH 5.5, 3 h; pH 5.5, 6 h; pH 6.0, 3 h; and pH 6.0, 6 h) and survival rate after further acid lethal treatment (pH 3.0, 20min) were measured. Bacteria were adjusted to OD₆₀₀ nm of 0.4 for counting. The error bars indicate the standard deviations from three independent replicates. Statistical significance was compared to the group of pH 5.5, 3 h: ns, no significant, ****, P ˂ 0.0001

Fig. 2

Characterization of constitutive promoters via RNA-seq. a Scatter-plot of gene expression level at pH 5.5 and 7.4 via RNA-seq. The red spots indicate significantly up-regulated genes; the green spots indicate significantly down-regulated genes; black spots indicate nonsignificant genes. b Venn diagram of the number of the top 2.0% of the most highly expressed genes under two conditions by RNA-seq. c Schematic diagram of the promoter-GFP cassette for promoter screening. The sequence of the promoter-GFP cassette is highlighted in red color. Fluorescence intensity of GFP by different constitutive promoters in wild-type EGD-e at pH 7.4 (d) and 5.5 (e), respectively. The error bars indicate the standard deviations from three independent replicates

Fig. 3

Fluorescent tracer of L. monocytogenes in vitro and in vivo. a Fluorescent tracer of L. monocytogenes in macrophage RAW264.7. b Fluorescent tracer of L. monocytogenes in vivo

Fig. 4

Measurement of UreB production. a Measurement of UreB production in EGD-eΔactA/inlB by western blotting. b Quantitative determination of UreB production based on the gray scan. The error bars indicate the standard deviations from three independent replicates. Statistical significance was compared to the group of P_hly: ns, no significant; *, P ˂ 0.05; **, P ˂ 0.01; ****, P ˂ 0.0001