Yersinia enterocolitica induces apoptosis in macrophages by a process requiring functional type III secretion and translocation mechanisms and involving YopP, presumably acting as an effector protein

Abstract

Yersiniae, causative agents of plague and gastrointestinal diseases, secrete and translocate Yop effector proteins into the cytosol of macrophages, leading to disruption of host defense mechanisms. It is shown in this report that Yersinia enterocolitica induces apoptosis in macrophages and that this effect depends on YopP. Functional secretion and translocation mechanisms are required for YopP to act, strongly suggesting that this protein exerts its effect intracellularly, after translocation into the macrophages. YopP shows a high level of sequence similarity with AvrRxv, an avirulence protein from Xanthomonas campestris, a plant pathogen that induces programmed cell death in plant cells. This indicates possible similarities between the strategies used by pathogenic bacteria to elicit programmed cell death in both plant and animal hosts.

Figure 1

A Y. enterocolitica Yop effector induces apoptosis in macrophages. (A) Percentage of TUNEL-positive nuclei in J774A.1 cells 4 h postinfection. n values refer to the number of total cells evaluated per treatment. All strains were tested a minimum of three times. WT, wild-type. Nonpolar mutations in genes required for secretion (yscN) and translocation (yopB, yopD and sycD) were confirmed by 12% SDS/PAGE (lanes correspond with mutants indicated above the gel). Arrowheads indicate the position of the deleted protein. The correct positions of Yops are indicated at left. (B) Phase contrast (left) and fluorescence (right) images depicting the results of the infection (cell morphology) and TUNEL reaction (nuclear morphology), respectively. (A) Uninfected cells, (B) wild-type E40, (C) yscN mutant, and (D) yopB mutant. Arrowheads indicate J774A.1 membrane blebbing induced by wild-type E40 infection.

Figure 2

YopP, a new Y. enterocolitica Yop effector, is required to induce apoptosis in macrophages. (A) 12% SDS/PAGE of secreted proteins from isogenic nonpolar yop mutants and overexpression of YopP in the yopP mutant. Lanes: 1, wild-type E40; 2, yopE; 3, yopH; 4, yopO; 5, yopM; 6, yopP; and 7, yopP+++ (yopP mutant overexpressing YopP). Arrowheads indicate the position of the deleted protein. Asterisk (∗) denotes overexpressed YopP. To clearly show the secretion profile of the yopO mutant, samples were overloaded with supernatants of (lane 8) wild-type E40 and (lane 9) yopO mutant cultures. Loading was based on equivalent numbers of bacteria. Molecular masses are shown at left, and positions of Yops are shown at right. (B) Percentage of TUNEL-positive nuclei in J774A.1 cells infected with wild-type strain E40 or isogenic nonpolar Yop effector mutants (4 h postinfection). n values refer to the number of total cells evaluated per treatment. All strains were tested a minimum of three times. (C) Percentage of TUNEL-positive nuclei in J774A.1 cells infected with wild-type (WT) E40, yopP mutant, or yopP+++. Error bars represent the SEM for 1 field of view (≈100–150 cells) from three separate coverslips. (D) Phase contrast (left) and fluorescence (right) images depicting the results of the infection (cell morphology) and TUNEL reaction (nuclear morphology), respectively in J774A.1 cells infected with (A) yopP mutant and (B) yopP⁺⁺⁺.

Figure 3

Extracellular Y. enterocolitica induce apoptosis. J774A.1 cells were infected in the presence or absence of 2.5 μg/ml cytochalasin D and processed for the TUNEL reaction at 4 h postinfection. n values indicate the total number of cells evaluated per treatment. Wild-type strain E40 and the yopE mutant were able to induce apoptosis as efficiently in the absence (A) or presence (B) of cytochalasin D (inhibitor of phagocytic bacterial uptake). Uninfected cells, and yscN, yopB, and yopP mutant-infected cells, were negative for the TUNEL reaction. All strains were tested a minimum of three times.

Figure 4

DNA fragmentation of Y. enterocolitica-infected J774A.1 cells. Oligonucleosomal length DNA fragmentation was observed in cells infected with wild-type (WT) and P+ (yopP+++) but not in noninfected (ni) cells or cells infected with P- (yopP mutant). J774A.1 cells were infected as described in Materials and Methods using a multiplicity of infection (moi) of either 10 or 100 bacteria per cell as indicated. J774A.1 DNA was isolated 2 h after infection and subjected to agarose gel electrophoresis (18). Molecular weights (in base pairs) are indicated at left.

Figure 5

Structural evidence for Y. enterocolitica-induced apoptosis. Semi-thin sections were stained with toluidine blue and examined by light microscopy (a–d). (a) Wild-type strain E40; apoptotic nuclei (arrows) and cell surface-associated bacteria (brown particles, arrowhead). (b) yscN secretion mutant; no apoptotic cells are detected. Abundance of internalized bacteria either in tight (single arrowhead) or spacious phagosomes (double arrowhead). (c) yopP effector mutant; no apoptotic cells are detected. Bacteria at the cell surface (arrowhead). (d) yopP⁺⁺⁺, apoptotic cells (arrows). Ultrastructural analysis of cells infected with yopP⁺⁺⁺ from d is shown in e and f. Typical features of apoptosis include (i) peripheral chromatin condensation in crescents, except in the vicinity of nuclear pores (large arrows); (ii) bulging of nuclear crescents into the cytoplasm, best seen in e; and (iii) appearance of central clusters of small particles of unknown nature, typical of apoptosis (small arrow at f). Nuclear and plasma membrane alterations contrast with a good ultrastructural preservation of cytoplasm, particularly of endoplasmic reticulum and mitochondria. (a–d, Bar = 10 μm; e–f, Bar = 2 μm).

Figure 6

Alignment between YopP (Y. enterocolitica), YopJ (Y. pseudotuberculosis), and avirulence protein AvrRxv from X. campestris. Identical amino acids are in red, and conserved amino acids are in blue.