An M29 Aminopeptidase from Listeria Monocytogenes Contributes to In Vitro Bacterial Growth but not to Intracellular Infection

Abstract

Aminopeptidases that catalyze the removal of N-terminal residues from polypeptides or proteins are crucial for physiological processes. Here, we explore the biological functions of an M29 family aminopeptidase II from Listeria monocytogenes (LmAmpII). We show that LmAmpII contains a conserved catalytic motif (EEHYHD) that is essential for its enzymatic activity and LmAmpII has a substrate preference for arginine and leucine. Studies on biological roles indicate that LmAmpII is required for in vitro growth in a chemically defined medium for optimal growth of L. monocytogenes but is not required for bacterial intracellular infection in epithelial cells and macrophages, as well as cell-to-cell spreading in fibroblasts. Moreover, LmAmpII is found as dispensable for bacterial pathogenicity in mice. Taken together, we conclude that LmAmpII, an M29 family aminopeptidase, can efficiently hydrolyze a wide range of substrates and is required for in vitro bacterial growth, which lays a foundation for in-depth investigations of aminopeptidases as potential targets to defend Listeria infection.

Keywords: Listeria monocytogenes; aminopeptidase; bacterial growth; infection; the M29 family.

Figure 1

The cytoplasmic putative aminopeptidase II (LmAmpII) is an active aminopeptidase II of the M29 family with a wide range of substrate specificities. (A) Localization of LmAmpII in L. monocytogenes. Proteins were separated through a 12% SDS-PAGE and immunoblotted with α- LmAmpII, α-InlB, α-LLO, or α-GAPDH antisera. The predicted molecular weight of each protein is indicated on the left. (B) SDS-PAGE analysis of the recombinant LmAmpII and its mutant variants with single site-directed mutagenesis at the predicted active sites. (C,D) Enzymatic activity of LmAmpII using Lys-, Arg-, Asp-, Asn-, Ser-, Gly-, Leu-, Phe-, Val-, or Ala-pNA and polypeptides Gly-Pro-, Ala-Ala-Pro-Phe-, D-Phe-Pip-Arg-, or Val-Gly-Gly-pNA as substrate at a concentration of 1 mM in 25 mM Tris-HCl (pH 7.4) buffer. (E) Aminopeptidase activity of the wild-type LmAmpII and its mutant variants (E250A, E316A, H345A, Y352A, H378A, and D380A) using Leu-pNA as a substrate in 25 mM Tris-HCl (pH 7.4) buffer. Data shown represent mean ± SD.

Figure 1

The cytoplasmic putative aminopeptidase II (LmAmpII) is an active aminopeptidase II of the M29 family with a wide range of substrate specificities. (A) Localization of LmAmpII in L. monocytogenes. Proteins were separated through a 12% SDS-PAGE and immunoblotted with α- LmAmpII, α-InlB, α-LLO, or α-GAPDH antisera. The predicted molecular weight of each protein is indicated on the left. (B) SDS-PAGE analysis of the recombinant LmAmpII and its mutant variants with single site-directed mutagenesis at the predicted active sites. (C,D) Enzymatic activity of LmAmpII using Lys-, Arg-, Asp-, Asn-, Ser-, Gly-, Leu-, Phe-, Val-, or Ala-pNA and polypeptides Gly-Pro-, Ala-Ala-Pro-Phe-, D-Phe-Pip-Arg-, or Val-Gly-Gly-pNA as substrate at a concentration of 1 mM in 25 mM Tris-HCl (pH 7.4) buffer. (E) Aminopeptidase activity of the wild-type LmAmpII and its mutant variants (E250A, E316A, H345A, Y352A, H378A, and D380A) using Leu-pNA as a substrate in 25 mM Tris-HCl (pH 7.4) buffer. Data shown represent mean ± SD.

Figure 2

Biochemical characterization of LmAmpII. (A) Effect of pH on aminopeptidase activity. The enzymatic assays were performed over a pH range from 3.5 to 11.5 in 25 mM Tris-HCl buffer using Leu-p-nitroaniline (Leu-pNA) as a substrate. (B) Effect of temperature on aminopeptidase activity. The enzymatic assays were performed in 25 mM Tris-HCl (pH 7.4) buffer over a temperature range from 4–50 °C using Leu-pNA as a substrate. (C) Effect of metal ions on aminopeptidase activity. Activity was assayed in the presence of different metal ions (Co²⁺, Cd²⁺, Zn²⁺, Mg²⁺, Mn²⁺, or Fe³⁺, each at 0.5 mM) using Leu-pNA as a substrate. (D) Effect of Co²⁺ on aminopeptidase activity against the inert polypeptide substrates. Activity was assayed in the presence of using the polypeptides Gly-Pro-, Ala-Ala-Pro-Phe-, D-Phe-Pip-Arg-, or Val-Gly-Gly-pNA as a substrate at a concentration of 1 mM in 25 mM Tris-HCl (pH 7.4) buffer. Data shown represent mean ± SD. The red lines in (A), (B), and (C) indicate 100% relative enzymatic activity.

Figure 3

The Michaelis–Menten kinetic parameters of LmAmpII. The Michaelis–Menten curves of LmAmpII were plotted, and the K_m, V_max, K_cat, and K_cat/K_m values were determined for each substrate. (A) Arg-pNA, (B) Leu-pNA, (C) Lys-pNA, (D) Ala-pNA, or (E) Phe-pNA. These assays were carried out using the specific substrate at a concentration range of 0–1000 μM in 25 mM Tris-HCl (pH 7.4), containing 0.05 μM enzyme. Data of each black dot shown represent mean ± SD.

Figure 4

LmAmpII is required for bacterial growth in a chemically defined medium for optimal growth of L. monocytogenes, but not for intracellular infection and virulence. (A) Construction of the LmAmpII deletion and complemented mutants, and protein expression was detected by Western blotting. (B,C) Growth curves of L. monocytogenes bacteria in the brain heart infusion (BHI) medium (B) and chemically defined medium (C). (D,E) Intracellular growth of L. monocytogenes in murine-derived Raw264.7 (D) and J774A.1 (E) macrophages. Gentamicin (50 μg/mL) was added 30 min post-infection. Cells infected with L. monocytogenes strains that were lysed at the indicated time points (2, 6, and 12 h), and viable bacteria were numbered by serial plating on BHI agar plates. (F) Adhesion, invasion, and proliferation of L. monocytogenes in human intestinal epithelial cells Caco-2. Cells infected with bacteria were lysed at the indicated time points, and viable bacteria were serially plated on BHI agar plates. The number of recovered bacteria shown that were able to invade cells and survive represent the mean ± SD for each strain. ns, not significant.

Figure 5

LmAmpII was not essential for L. monocytogenes virulence in the mouse model. (A) Plaque assay performed on L929 fibroblast monolayers infected by L. monocytogenes. The plaque numbers of the mutant strains were indicated as a percentage of those formed by the wild-type strain. (B) Proliferation of L. monocytogenes in mice organs. Mice were inoculated intraperitoneally with 2 × 10⁶ CFU bacteria, and animals were euthanized 24 and 48 h post-infection (h.p.i), and numbers of bacteria colonized in organs shown represent the mean ± SD of the log₁₀cfu per organ for each group. (C) Kaplan–Meier survival of the infected ICR mice over time. The mice were challenged intraperitoneally with 2 × 10⁶ CFU bacteria and monitored for up to 7 days post-inoculation. Data shown represented as the percentage survival over time and significance was determined via a Log-rank test. ns, not significant.