A crucial role for profilin-actin in the intracellular motility of Listeria monocytogenes

Abstract

We have examined the effect of covalently crosslinked profilin-actin (PxA), which closely matches the biochemical properties of ordinary profilin-actin and interferes with actin polymerization in vitro and in vivo, on Listeria monocytogenes motility. PxA caused a marked reduction in bacterial motility, which was accompanied by the detachment of bacterial tails. The effect of PxA was dependent on its binding to proline-rich sequences, as shown by the inability of PH133SxA, which cannot interact with such sequences, to impair Listeria motility. PxA did not alter the motility of a Listeria mutant that is unable to recruit Ena (Enabled)/VASP (vasodilator-stimulated phosphoprotein) proteins and profilin to its surface. Finally, PxA did not block the initiation of actin-tail formation, indicating that profilin-actin is only required for the elongation of actin filaments at the bacterial surface. Our findings provide further evidence that profilin-actin is important for actin-based processes, and show that it has a key function in Listeria motility.

Figure 1

Profilin–actin impairs intracellular Listeria monocytogenes motility. PtK2 cells infected with L. monocytogenes were injected with 5 mg ml⁻¹ profilin–actin (PxA; needle concentration). Oregon 'Green dextran was included in the injection mixture to identify injected cells. Forty minutes after injection, PtK2 cells were fixed and stained with Texas-Red-conjugated phalloidin. The insets in (A) are enlarged in (B) and (C) In uninjected cells, Listeria were associated with normal actin tails (inset (C) in (A), and arrows in (C)), whereas in cells that received PxA, the bacteria were associated with short tails (inset (B) in (A), and arrows in (B)). Scale bars, 10 μm.

Figure 2

Effect of profilin–actin on Listeria motility. (A) Profilin–actin (PxA) reduces Listeria monocytogenes speed and causes actin-tail detachment. Listeria-infected PtK2 cells were injected with 20 mg ml⁻¹ PxA and were observed by video microscopy. Before injection, Listeria were associated with normal actin tails and moved at an average speed of 10.2 ± 2.58 μm min⁻¹ (arrows in frames for −230 s and −130 s). The injection of PxA caused an impairment of Listeria motility, followed by the rapid disassembly of the actin tails (arrowheads in frames for +130 s and +180 s). Three minutes after injection of PxA, Listeria cells were associated with a fuzzy, phase-dense material, and their movement became slow and irregular. (B,C) Effect of PxA on the instantaneous (B) and average (C) speed of Listeria. The injection of 5 or 20 mg ml⁻¹ PxA caused a decrease in bacterial motility, whereas the injection of G-buffer had no effect. The origin of the axes corresponds to injection time in (B). Error bars in (C) represent one standard deviation above the mean. Numbers in brackets in (C) indicate the number of motile bacteria that were examined. Scale bar, 10 μm.

Figure 3

Effects of profilin–actin on Listeria monocytogenes motility analysed in Madin–Darby canine kidney cells expressing green fluorescent protein–actin. MDCK cells that stably expressed green fluorescent protein (GFP)–actin were infected with Listeria and injected with 5 mg ml⁻¹ profilin–actin (PxA). Upper panels show phase-contrast images; lower panels show corresponding GFP–actin fluorescence. Shortly after injection, a break (green arrow; frame for 32 s) appeared between the bacterium (red arrowhead, lower frame for 32 s) and its tail, followed by the complete depolymerization of the actin tail (frames for 76 s and 120 s). The bacterium remained associated with a cloud of actin filaments (white arrows and arrowheads, frames for 76 s and 120 s) and moved slowly. Scale bar, 5 μm.

Figure 4

(A) The ability of profilin–actin to interact with proline-rich regions is essential for its inhibitory effect on Listeria monocytogenes motility. PtK2 cells infected with wild-type Listeria were injected with 5 mg ml⁻¹ P_H133SxA (a complex of the H133S mutant form of profilin with actin) and observed using video microscopy. Before injection, wild-type Listeria were associated with actin tails and moved at normal speed (arrow in frames for −190 s and −90 s), which was not affected by the injection of P_H133SxA (arrow in frames for +100 s and +200 s). (B,C) Quantification of Listeria speed after injection with profilin–actin (PxA) or P_H133SxA. The effects of PxA and P_H133SxA on the instantaneous (B) and average (C) speed of Listeria ActA5 and wild-type, respectively, are shown. The injection of 5 mg ml⁻¹ P_H133SxA or of G-buffer did not alter the motility of wild-type Listeria. Similarly, PxA did not change the motility of Listeria ActA5. The origin of the x axis in (B) corresponds to the time of injection. Error bars in (C) represent one standard deviation above the mean. Numbers in brackets in (C) indicate the number of motile bacteria examined.

Figure 5

Profilin–actin does not inhibit the initiation of Listeria monocytogenes actin tails in mouse cytosolic brain extracts. Mouse cytosolic brain extracts were incubated with various concentrations of profilin–actin (PxA) on ice for 30 min. Bacteria were then added to the mixture, which was incubated for 15 min at 20 °C. In control extracts, Listeria induced the formation of actin tails and moved at an average speed of 0.5 ± 0.16 μm min⁻¹ (arrows in (A,C,E); (G)). PxA induced a concentration-dependent reduction of bacterial motility (arrows in (B,D,F); (G)), but did not affect the initiation of actin tails. Note that bacteria occasionally aggregated during centrifugation before incubation with the extract. This led to movement diverging from a central point, which is clearly visible in (B,D,F). Scale bar, 10 μm. (G) Box and whiskers plots of bacterial speed. Filled circles indicate the mean; lines in the middle of the boxes indicate the median; the tops of the boxes indicate the 75th quartile and the bottoms of the boxes indicate the 25th quartile; 'whiskers' indicates the 10th and 90th percentiles, respectively.