The sortase SrtA of Listeria monocytogenes is involved in processing of internalin and in virulence

Abstract

Listeria monocytogenes is an intracellular gram-positive human pathogen that invades eucaryotic cells. Among the surface-exposed proteins playing a role in this invasive process, internalin belongs to the family of LPXTG proteins, which are known to be covalently linked to the bacterial cell wall in gram-positive bacteria. Recently, it has been shown in Staphylococcus aureus that the covalent anchoring of protein A, a typical LPXTG protein, is due to a cysteine protease, named sortase, required for bacterial virulence. Here, we identified in silico from the genome of L. monocytogenes a gene, designated srtA, encoding a sortase homologue. The role of this previously unknown sortase was studied by constructing a sortase knockout mutant. Internalin was used as a reporter protein to study the effects of the srtA mutation on cell wall anchoring of this LPXTG protein in L. monocytogenes. We show that the srtA mutant (i) is affected in the display of internalin at the bacterial surface, (ii) is significantly less invasive in vitro, and (iii) is attenuated in its virulence in the mouse. These results demonstrate that srtA of L. monocytogenes acts as a sortase and plays a role in the pathogenicity.

FIG. 1.

Alignment of L. monocytogenes SrtA with S. aureus sortase. Alignment was performed using CLUSTALw. Identical residues are boxed. L. monocytogenes SrtA comprises 222 amino acids; the sortase of S. aureus comprises 206 amino acids.

FIG. 2.

Western blot analysis of internalin in culture supernatants of BHI-grown cells. (A) Supernatants were harvested at early (OD₆₀₀ = 0.4), log (OD₆₀₀ = 0.6), late log (OD₆₀₀ = 0.8), and stationary (Stat) phase from EGD (WT) and EGDΔsrtA (ΔS). Loading corresponds to 100 μl of bacterial culture (adjusted to a final OD₆₀₀ of 1). Internalin was detected with MAb L7.7 at a final dilution of 1/1,000 (indicated by a black arrowhead). (B) Supernatants were harvested at stationary phase from EGD (WT), EGDΔsrtA (ΔS), EGDΔsrtA::aphA3 (ΔS-K), and EGDΔsrtA complemented with the wild-type srtA gene (Comp). Internalin was detected with MAb K18.4 at a final dilution of 1/1,000. Loading corresponds to 100 μl of bacterial culture (adjusted to a final OD₆₀₀ of 1). Molecular mass markers are indicated in kilodaltons to the left of the panels.

FIG. 3.

Western blot analysis of internalin in culture supernatants of RPMI-grown cells. (A) Detection with MAb L7.7. (B) Detection with MAb C20.4. Three major forms of internalin protein were detected by MAb L7.7 in the culture supernatant of the srtA mutant. The upper bands detected in the mutant are indicated by open arrowheads. The two forms with a lower apparent molecular mass than that of internalin released by the wild-type strain are indicated by arrows. Both polypeptides have the same N terminus as the mature form of wild-type internalin (ATITQ). Internalin was detected with MAb L7.7 or MAb C20.4 at final dilutions of 1/1,000. Molecular mass markers are indicated in kilodaltons to the left of the panels. ΔS, EGDΔsrtA; WT, EGD.

FIG. 4.

Western blot analysis of SDS-treated bacteria. In the srtA mutant (ΔS), essentially all internalin was solubilized by SDS treatment, while in the wild-type (WT) strain, only a fraction of internalin was solubilized. (A) The uppermost form of internalin released by the mutant strain is indicated by an arrow. Internalin was detected with MAb L7.7 at a final dilution of 1/1,000. (B) In the wild-type strain, as well as in the srtA mutant, the membrane-anchored protein ActA is as good as totally released in the supernatant after incubation of the bacteria in hot SDS. ActA was detected with anti-ActA polyclonal serum at a final dilution of 1/1,000. Molecular mass markers are indicated in kilodaltons to the left of the panels. Abbreviations: S, soluble fraction; p, pellet fraction.

FIG. 5.

Invasivity assay of EGD and ΔsrtA into Caco-2 and HepG-2 cells. Invasiveness was evaluated in two different types of cell lines: Caco-2 cells (A) and HepG-2 cells (B). Cell monolayers were incubated for 1 h at 37°C with approximately 100 bacteria per cell. After washing, the cells were reincubated for 4 h in fresh culture medium containing gentamicin (10 mg liter⁻¹). At 2 and 4 h, the cells were washed again and lysed and viable bacteria were counted on BHI plates. The percentage of entry is the number of bacteria that survived in the presence of gentamicin per number of inoculated bacteria. Values are means of three different experiments. Error bars show standard deviations. Abbreviations: WT, EGD; ΔS, EGDΔsrtA; ΔS-K, EGDΔsrtA::aphA3; Comp, EGDΔsrtA complemented with the wild-type srtA gene.

FIG. 6.

In vivo survival of the ΔsrtA mutant. The kinetics of bacterial growth was monitored in organs of mice infected with the srtA mutant, compared with EGD as a positive control. Mice were inoculated with 10⁶ bacteria. Bacterial survival was monitored in the spleen (A), liver (B), and brain and blood (C) over a 7-day period. The counts in the blood for the srtA mutant are represented by dashed lines and black diamonds. Symbols: ▪, EGD; □, ΔsrtA. ▪†, death.