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Apoptosis of murine peritoneal macrophages induced by an avian pathogenic strain of Escherichia coli
FEMS Microbiology Letters 179 (1999) 73^78
Apoptosis of murine peritoneal macrophages induced by an avian pathogenic strain of Escherichia coli V.S. Rodrigues a , M.C. Vidotto a , I. Felipe a , D.S. Santos b , L.C.J. Gaziri
c;
*
a
Departamento de Patologia Geral, Universidade Estadual de Londrina, Londrina, Brazil Instituto de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Departamento de Cieªncias Fisiolo¨gicas, Universidade Estadual de Londrina, 86051-990 Londrina, Brazil b
c
Received 18 January 1999; received in revised form 12 July 1999; accepted 20 July 1999
Abstract The mechanisms used by avian strains of Escherichia coli to invade the respiratory epithelia, leading to septicemia in poultry, are not well-established. In this work, we show that resident murine peritoneal macrophages infected in vitro with an avian strain of E. coli underwent apoptosis 4 h after infection (55.6% of apoptosis in infected cells versus 3.5% in non-infected cells). Heat-inactivated bacteria did not induce apoptosis and the inhibition of phagocytosis by pretreatment of cells with cytochalasin D reduced the number of apoptotic cells from 55.6 to 13.9% (P 6 0.05), showing that the bacteria must be intracellularly located and viable to induce apoptosis. Therefore, these data suggest that induction of macrophage apoptosis may be a pathogenic mechanism employed by avian E. coli to circumvent the host defences and invade the respiratory epithelia. ß 1999 Published by Elsevier Science B.V. All rights reserved. Keywords : Macrophage; Apoptosis ; Colicin V; Escherichia coli
1. Introduction Avian septicemic strains of E. coli cause upper respiratory infections in chickens, which commonly develop into septicemia and death, causing important worldwide economic losses in the poultry industry [1]. Some ¢mbriae which mediate the adhesion of avian E. coli to tracheal cells have been identi¢ed [2,3], but the mechanisms employed by those bacteria to breach through barriers such as the respiratory
* Corresponding author. Tel.: +55 (43) 3714307; Fax: +55 (43) 3714207.
epithelia are not established. Toxins produced by E. coli might contribute through di¡erent mechanisms to its invasiveness. For instance, hemolysin forms pores in the membrane of target cells [4] and causes the release of mediators from in£ammatory cells [5]. Production of colicin V by E. coli has been correlated with its invasiveness and pathogenicity [6,7]. Its possible role as a virulence factor is unclear, although it is known that colicins act as poreformers that disrupt the membrane potential of target cells [8^10], suggesting that colicin V might damage the plasma membrane or the internal membranes of phagocytic cells. Induction of programmed cell death or apoptosis by bacteria may constitute an important factor in
0378-1097 / 99 / $20.00 ß 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 3 7 5 - 4
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pathogenesis, since cell death, particularly death of phagocytes, might favor bacterial invasion [11^13]. Following the initial demonstration that Shigella £exneri induces macrophage apoptosis in vitro [14], other bacteria [15^18], as well as bacterial proteins [19^21], were also shown to induce apoptosis. A fortuitous observation from our laboratory suggested that a colicin V-producing strain of septicemic avian E. coli killed murine peritoneal macrophages in vitro and that some of the macrophages' nuclei remnants were apoptotic. In this study, therefore, we have investigated whether avian septicemic E. coli induces macrophage apoptosis in vitro and whether this hypothetical activity correlates with the capability of bacteria to produce colicin V.
2. Materials and methods 2.1. Bacteria and growth conditions A colicin V-producing strain of E. coli (UEL 17), isolated from a colisepticemic chicken [7], and one of its Col V3 isogenic derivatives were used in this study. The bacteria were grown to the exponential phase (D.O. = 1U109 ) in Luria-Bertani broth at 37³C with aeration and then inoculated into the medium used for the phagocytosis assay (RPMI 1640, Sigma) and grown overnight. Three dilutions of each culture were plated on agar for determination of the number of colony forming units ml31 . S. £exneri M90T (kindly supplied by P.J. Sansonetti) was grown under similar conditions and used as a positive control for induction of apoptosis. 2.2. Phagocytic cells Resident peritoneal phagocytes were collected from Swiss mice (weighing 28^32 g) by rinsing the peritoneal cavity of each animal with 3 ml of RPMI medium containing albumin (1%). The cells were cytospun, resuspended in RPMI medium and counted in a hemocytometer. For phagocytosis assays, the cells were allowed to adhere to coverslips for 1 h at 37³C and the monolayers formed were rinsed in RPMI medium and incubated with the bacteria (100 bacteria per phagocyte). Cell viability was estimated by trypan blue exclusion [22].
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2.3. Apoptosis assay Microscopic detection of apoptosis [23] was carried out both by identi¢cation of cells bearing condensed and fragmented nuclei in HE-stained preparations and by an in situ terminal deoxynucleotidyl transferase nick end labelling assay (TUNEL). For both assays, monolayers of phagocytic cells were incubated with bacteria for 30 min in RPMI medium at 37³C, the monolayers were then washed and incubated for 15 min with gentamicin (100Wg ml31 in RPMI medium) to remove non-ingested bacteria [24] and re-incubated in RPMI to complete 4 h of incubation and ¢nally ¢xed in absolute methanol. The TUNEL assay was carried out as follows. The cell monolayers were incubated at 37³C for 1 h in a medium containing 0.02 M 5-bromo-2P-deoxyuridine (BrdU), 30 U of terminal deoxynucleotidyl transferase (TdT), 0.1 M sodium cacodylate, 2 mM CoCl2 , 50 Wg ml31 BSA, 30 mM Tris-HCl and 4 mM of each dCTP, dGTP and dATP (Sigma). After the hybridization incubation, the preparations were rinsed with 50% formamide, then rinsed three times with 0.3 M NaCl containing 0.03 M sodium citrate, followed by three rinses in phosphate-bu¡ered saline (PBS) containing 0.05% Nonidet P-40 (NP bu¡er). Mouse anti-BrdU antibody (Sigma) diluted 1:200 in PBS was then applied over the cell monolayers for 30 min, the preparations were rinsed three times in NP bu¡er and incubated for 30 min with anti-mouse secondary antibody conjugated with FITC (Sigma). The preparations were analyzed in a £uorescence microscope (Zeiss) using a FITC ¢lter cube, background labelling was assessed on duplicate preparations incubated without TdT. 2.4. Analysis of DNA fragmentation DNA was extracted by the phenol-chloroform method [25] and fractionated in a 1% agarose gel in Tris-borate-EDTA bu¡er. Brie£y, 106 peritoneal phagocytic cells were co-incubated with 108 bacteria for 30 min, 100 Wg ml31 of gentamicin was added to kill extracellular bacteria and after 10 min, the cells were pelleted and resuspended in RPMI medium and re-incubated to complete 4 h. Control preparations were similarly processed, but with no addition of the bacteria. The cells were then pelleted, treated with
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the lysis bu¡er (10 mM Tris-HCl, 10 mM EDTA, 0.5% sodium dodecyl sulfate) and incubated at 56³C for 2 h. This preparation was treated with 100 Wg of RNAse A for 30 min at 37³C and the DNA was extracted with phenol-chloroform, precipitated overnight at 320³C in 5 M NaCl and pelleted with ethanol. The pellet was resuspended in 30 Wl of Tris-EDTA bu¡er and fractionated in a 1% agarose gel. The DNA was stained with ethidium bromide and visualized and photographed on an Image Master (Pharmacia).
3. Results 3.1. E. coli (UEL 17) induces macrophage apoptosis in vitro Macrophages infected with E. coli (UEL 17) presented the nuclear condensation and fragmentation pattern characteristic of apoptosis (Fig. 1B). Samples of the same macrophage population incubated with no bacteria presented normal morphology (Fig. 1A). DNA extracted from phagocytes incubated with E. coli showed a ladder pattern characteristic of apoptosis on agarose gel electrophoresis (Fig. 2). Fluorescence of the nuclei of infected cells, which reacted with BrdU on TUNEL assays, shows apoptotic cells (Fig. 3B), whereas the nuclei of non-infected cells did not react with the £uorescent FITC-conjugated antibody (Fig. 3A). Macrophages infected with S. £exneri, used as a positive control of apoptosis, showed a £uorescence pattern (Fig. 3D) similar to that preTable 1 E¡ect of cytochalasin D on macrophage apoptosis induced by E. coli Condition
No bacteria added E. coli S. £exneri
Apoptotic macrophages (%) Control
Cytochalasin D
3.5 þ 1.9 55.6 þ 1.4 42.5 þ 2.1
3.2 þ 1.9 13.1 þ 9.5 15.5 þ 2.0
The percentage of macrophage apoptosis was determined 4 h after infection with the bacteria, v 100 HE-stained macrophages were scored for the presence of apoptosis. S. £exneri was used as a positive control of apoptosis. Values are means þ S.D. of at least three independent experiments. P 6 0.05 for infected macrophages untreated versus treated with cytochalasin D.
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Fig. 1. Morphology of murine peritoneal macrophages infected with E. coli (UEL 17). Macrophages were either incubated with no bacteria (A) or incubated with viable (B) or heat-inactivated E. coli (C). Arrows on C point to examples of macrophages containing bacteria. After 4 h of incubation, the cells were stained with HE (100 bacteria per cell, magni¢cation U1000).
sented by the cells infected with E. coli. About 55% of macrophages were apoptotic 4 h after infection with E. coli (Table 1).
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3.2. Heat-inactivated E. coli does not induce macrophage apoptosis Macrophages co-incubated with heat-inactivated E. coli did not show the nuclear condensation and fragmentation characteristic of apoptosis (Fig. 1C). DNA extracted from cells incubated with heat-inactivated bacteria did not show the ladder pattern of fragmentation on agarose gel electrophoresis, remaining on the top of the gel (electrophoretogram not shown). That the heat-inactivated bacteria were phagocytosed is suggested by the presence of many bacteria surrounded by a clear halo within the contours of the macrophages (Fig. 1C). Similar preparations stained with acridine orange show, under the UV microscope, that many red (dead) bacteria are clustered inside the macrophages and segregated outside the nuclear region (UV micrograph not shown). 3.3. Inhibition of phagocytosis by cytochalasin D abolishes apoptosis induced by E. coli Macrophages pre-incubated with cytochalasin D (2 Wg ml31 ) for 30 min and then infected with E. coli did not react with the £uorescent tag on TUNEL assays (Fig. 3C). On optical microscopy of HEstained preparations, the nuclei of macrophages pretreated with cytochalasin D and infected with E. coli were not condensed or fragmented (micrograph not shown), the cell morphology being indistinguishable from that of normal macrophages shown on Fig. 1A. Pretreatment of macrophages with cytochalasin D reduced the percentage of apoptotic cells (Table 1) from 55.6 þ 1.4 to 13.1 þ 9.5 (S.D.). 3.4. Both the colicin V-producing strain E. coli UEL 17 and an isogenic Col V3 derivative induce macrophage apoptosis The wild-type E. coli UEL 17 used in the experiments described above carries three large plasmids, one of which encodes colicin V. An isogenic strain which lacks this plasmid (E. coli UEL 17 Col V3 ) was also capable of inducing apoptosis, as indicated by analysis of DNA fragmentation (Fig. 2). Macrophages infected with the Col V3 strain for 30 min and then re-incubated to complete 4 h of incubation presented nuclear fragmentation and condensation
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Fig. 2. DNA fragmentation of murine peritoneal macrophages infected with E. coli (UEL 17). Cells were co-incubated with bacteria for 30 min, treated with gentamicin (100 Wg ml31 ) and cytospun and re-incubated (4 h total). Lanes: (A) molecular mass markers; (B, C) controls, non-infected; (D, E) cells infected with E. coli Col V ; (F, G) cells infected with isogenic E. coli Col V3 .
(micrograph not shown) similar to that caused by the Col V strain shown on Fig. 1B.
4. Discussion Avian pathogenic strains of E. coli can cause upper respiratory infections that spread to the lower respiratory tract and subsequently develop into colisepticemia in poultry. The mechanisms and virulence factors employed by those bacteria to circumvent the host defences and cross through the epithelial barriers are not completely known. In this study, we observed that a strain of avian E. coli caused macrophage apoptosis in vitro, which suggests that induction of apoptosis might favor the invasiveness of those bacteria. That the mechanism of macrophage death was predominantly apoptosis was shown by the identi¢cation of nuclear fragmentation and condensation by optical microscopy, by in situ analysis of DNA fragmentation by TUNEL assays and by the presence of the characteristic ladder pattern of DNA-banding on agarose gel elec-
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Fig. 3. TUNEL reaction in infected murine peritoneal macrophages. Cells were incubated with E. coli for 30 min, washed with gentamicin (100 Wg ml31 ) to remove non-ingested bacteria and re-incubated to complete 4 h of incubation. TUNEL reaction was used to label 3P-OH ends with £uorescein. Cells were either non-infected (A) or infected (B) or pretreated with cytochalasin D for 30 min and then infected (C). Macrophages infected with S. £exneri M90T were used as a positive control of apoptosis (D). Bars represent 10 Wm.
trophoresis. The time-course of apoptosis induction by E. coli was similar to that reported for S. £exneri [14], Salmonella typhimurium [17] and Yersinia enterocolitica [18]. 1^2 h after co-incubation of E. coli with macrophages, apoptosis could already be detected (data not shown) and after 4 h of incubation, about 55% of macrophages were apoptotic. This relatively short time-course of apoptosis induction seems to be common to phagocytosed bacteria and to invasive bacteria, in contrast to the longer times taken for the induction of apoptosis by bacteria adapted to intracellular living, such as Mycobacterium tuberculosis [26]. Inhibition of phagocytosis by cytochalasin D prevented the induction of apoptosis by E. coli, indicating that the bacteria must be intracellularly located to induce apoptosis. Similar results were observed
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with S. £exneri [14] and S. typhimurium [17], whose ability to induce macrophage apoptosis is blocked by cytochalasin D. Moreover, heat-inactivated E. coli were phagocytosed but failed to induce apoptosis. Therefore, these results show that the induction of apoptosis by E. coli depends both on the bacteria being intracellularly located and viable. This requirement for bacterial viability suggests that some protein(s) secreted by E. coli mediate the induction of apoptosis, as has been observed for S. £exneri [19,21] and for Salmonella spp. [20]. Colicin V-producing strains of E. coli seem to be more virulent than non-producers [6,7]. A direct role of colicin V as a virulence factor has not been conclusively demonstrated, although it is known that colicins disrupt the membrane potential of target cells [8^10]. Because the insertion of colicins on their
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target membranes is known to depend on the potential di¡erence across those membranes and given that the phagolysosome is at a pH di¡erent from that of the cytoplasm, we hypothesized that colicin V secreted by phagocytosed E. coli could be inserted in the membrane of the phagosome and activate apoptosis. We found that both the colicin V-producing wild-type strain of E. coli and one isogenic Col V3 strain induced macrophage apoptosis, showing that the production of colicin V by E. coli is not necessary for its ability to induce apoptosis. The results of this study suggest, therefore, that the induction of macrophage apoptosis by avian septicemic E. coli might be a pathogenic mechanism employed by those bacteria to circumvent the host cellular defences and that to induce apoptosis, the bacteria must be both intracellularly located and viable. Moreover, these results also show that the production of colicin V by E. coli is not required for its ability to induce macrophage apoptosis.
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
References [1] Gross, W.B. (1984) Factors a¡ecting the development of respiratory disease complex in chickens. Avian Dis. 34, 607^ 610. [2] Dozois, C.M., Pourbakhsh, S.A. and Fairbrother, J.M. (1995) Expression of P and type 1 (F1) ¢mbriae in pathogenic Escherichia coli from poultry. Vet. Microbiol. 45, 297^309. [3] Vidotto, M.C., Navarro, H.R. and Gaziri, L.C.J. (1997) Adherence pili of pathogenic strains of avian Escherichia coli. Vet. Microbiol. 59, 79^87. [4] Menestrina, G., Mackman, N., Holland, I.B. and Bhakdi, S. (1987) Escherichia coli haemolysin forms voltage-dependent ion channels in lipid membranes. Biochem. Biophys. Acta 905, 109^117. [5] Ko«nig, B., Ludwig, A., Goebel, W. and Ko«nig, W. (1994) Pore formation by the Escherichia coli K-hemolysin: role for mediator release from human in£ammatory cells. Infect. Immun. 62, 4611^4617. [6] Smith, H.W. and Huggins, M.B. (1976) Further observations on the colicin V plasmid of Escherichia coli with pathogenicity and with survival in the alimentary tract. J. Gen. Microbiol. 92, 335^350. [7] Vidotto, M.C., Mu«ller, E.E., Freitas, J.C., Al¢eri, A.A., Guimara¬es, I.G. and Santos, D.S. (1990) Virulence factors of avian Escherichia coli. Avian Dis. 34, 531^538. [8] Yang, C.C. and Konisky, J. (1984) Colicin V treated Escherichia coli does not generate membrane potential. J. Bacteriol. 158, 757^759. [9] Slatin, S.L., Raymond, L. and Finkelstein, A. (1986) Gating
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[18]
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of a voltage-dependent channel (Colicin E1) in planar lipid bilayers : the role of protein translocation. J. Membr. Biol. 92, 247^254. Merril, A.R. and Cramer, W.A. (1990) Identi¢cation of a voltage-responsive segment of the potencial-gated colicin E1 ion channel. Biochemistry 29, 8529^8534. Guinchon, A. and Zychlinsky, A. (1996) Apoptosis as a trigger of in£ammation in Shigella-induced cell death. Biochem. Soc. Trans. 24, 1051^1054. Guinchon, A. and Zychlinsky, A. (1997) Clinical isolates of Shigella species induce apoptosis in macrophages. J. Infect. Dis. 175, 470^473. Finlay, B.B. and Cossart, P. (1997) Exploitation of mammalian host cell functions by bacterial pathogens. Science 276, 718^725. Zychlinsky, A., Pre¨vost, M.C. and Sansonetti, P.J. (1992) Shigella £exneri induces apoptosis in infected macrophages. Nature 358, 167^169. Khelef, N., Zychlinsky, A. and Guiso, N. (1993) Bordetella pertussis induces apoptosis in macrophages: role of adenylate cyclase-hemolysin. Infect. Immun. 61, 4064^4071. Baran, J., Guzik, K., Hryniewicz, W., Ernst, M., Flad, H.-D. and Pryjma, J. (1996) Apoptosis of monocytes and prolonged survival of granulocytes as a result of phagocytosis of bacteria. Infect. Immun. 64, 4242^4248. Monack, D.M., Raupach, B., Hromockyj, A.E. and Falkow, S. (1996) Salmonella typhimurium invasion induces apoptosis in infected macrophages. Proc. Natl. Acad. Sci. USA 93, 9833^9838. Ruckdeschel, K., Roggenkamp, A., Lafont, V., Mangeat, P., Heesemann, J. and Rouot, B. (1997) Interaction of Yersinia enterocolitica with macrophages leads to macrophage cell death through apoptosis. Infect. Immun. 65, 4813^4821. Zychlinsky, A., Kenny, B., Me¨nard, R., Pre¨vost, M.C., Holland, I.B. and Sansonetti, P.J. (1994) Ipa B mediates macrophage apoptosis induced by Shigella £exneri. Mol. Microbiol. 11, 619^627. Chen, L.M., Kaniga, K. and Galan, J.E. (1996) Salmonella spp. are cytotoxic for cultured macrophages. Mol. Microbiol. 21, 1101^1115. Chen, Y., Smith, M.R., Thirumalai, K. and Zychlinsky, A. (1996) A bacterial invasin induces macrophage apoptosis by binding directly to ICE. EMBO J. 15, 3853^3860. Harlow, E. and Lane, D. (1988) Antibodies: a Laboratory Manual, p. 256. Cold Spring Harbor Laboratory, NY. McCarthy, N.J. and Evan, G.I. (1998) Methods for detecting and quantifying apoptosis. Curr. Top. Dev. Biol. 36, 259^278. Vaudaux, P. and Waldvogel, F.A. (1979) Gentamicin antibacterial activity in the presence of human polymorphonuclear leukocytes. Antimicrob. Agents Chemother. 16, 743^749. Davis, L.G., Kuehl, W.M. and Battey, J.F. (1994) Basic Methods in Molecular Biology, pp. 16^21. Appleton and Lange, Norwalk, CT. Keane, J., Balcewicz-Sablinska, M.K., Remold, H.G., Chupp, G.L., Meek, B.B. and Kornfeld, H. (1997) Infection by Mycobacterium tuberculosis promotes human alveolar macrophages apoptosis. Infect. Immun. 65, 298^304.
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Apoptosis of murine peritoneal macrophages induced by an avian pathogenic strain of Escherichia coli V.S. Rodrigues a , M.C. Vidotto a , I. Felipe a , D.S. Santos b , L.C.J. Gaziri
c;
*
a
Departamento de Patologia Geral, Universidade Estadual de Londrina, Londrina, Brazil Instituto de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Departamento de Cieªncias Fisiolo¨gicas, Universidade Estadual de Londrina, 86051-990 Londrina, Brazil b
c
Received 18 January 1999; received in revised form 12 July 1999; accepted 20 July 1999
Abstract The mechanisms used by avian strains of Escherichia coli to invade the respiratory epithelia, leading to septicemia in poultry, are not well-established. In this work, we show that resident murine peritoneal macrophages infected in vitro with an avian strain of E. coli underwent apoptosis 4 h after infection (55.6% of apoptosis in infected cells versus 3.5% in non-infected cells). Heat-inactivated bacteria did not induce apoptosis and the inhibition of phagocytosis by pretreatment of cells with cytochalasin D reduced the number of apoptotic cells from 55.6 to 13.9% (P 6 0.05), showing that the bacteria must be intracellularly located and viable to induce apoptosis. Therefore, these data suggest that induction of macrophage apoptosis may be a pathogenic mechanism employed by avian E. coli to circumvent the host defences and invade the respiratory epithelia. ß 1999 Published by Elsevier Science B.V. All rights reserved. Keywords : Macrophage; Apoptosis ; Colicin V; Escherichia coli
1. Introduction Avian septicemic strains of E. coli cause upper respiratory infections in chickens, which commonly develop into septicemia and death, causing important worldwide economic losses in the poultry industry [1]. Some ¢mbriae which mediate the adhesion of avian E. coli to tracheal cells have been identi¢ed [2,3], but the mechanisms employed by those bacteria to breach through barriers such as the respiratory
* Corresponding author. Tel.: +55 (43) 3714307; Fax: +55 (43) 3714207.
epithelia are not established. Toxins produced by E. coli might contribute through di¡erent mechanisms to its invasiveness. For instance, hemolysin forms pores in the membrane of target cells [4] and causes the release of mediators from in£ammatory cells [5]. Production of colicin V by E. coli has been correlated with its invasiveness and pathogenicity [6,7]. Its possible role as a virulence factor is unclear, although it is known that colicins act as poreformers that disrupt the membrane potential of target cells [8^10], suggesting that colicin V might damage the plasma membrane or the internal membranes of phagocytic cells. Induction of programmed cell death or apoptosis by bacteria may constitute an important factor in
0378-1097 / 99 / $20.00 ß 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 3 7 5 - 4
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pathogenesis, since cell death, particularly death of phagocytes, might favor bacterial invasion [11^13]. Following the initial demonstration that Shigella £exneri induces macrophage apoptosis in vitro [14], other bacteria [15^18], as well as bacterial proteins [19^21], were also shown to induce apoptosis. A fortuitous observation from our laboratory suggested that a colicin V-producing strain of septicemic avian E. coli killed murine peritoneal macrophages in vitro and that some of the macrophages' nuclei remnants were apoptotic. In this study, therefore, we have investigated whether avian septicemic E. coli induces macrophage apoptosis in vitro and whether this hypothetical activity correlates with the capability of bacteria to produce colicin V.
2. Materials and methods 2.1. Bacteria and growth conditions A colicin V-producing strain of E. coli (UEL 17), isolated from a colisepticemic chicken [7], and one of its Col V3 isogenic derivatives were used in this study. The bacteria were grown to the exponential phase (D.O. = 1U109 ) in Luria-Bertani broth at 37³C with aeration and then inoculated into the medium used for the phagocytosis assay (RPMI 1640, Sigma) and grown overnight. Three dilutions of each culture were plated on agar for determination of the number of colony forming units ml31 . S. £exneri M90T (kindly supplied by P.J. Sansonetti) was grown under similar conditions and used as a positive control for induction of apoptosis. 2.2. Phagocytic cells Resident peritoneal phagocytes were collected from Swiss mice (weighing 28^32 g) by rinsing the peritoneal cavity of each animal with 3 ml of RPMI medium containing albumin (1%). The cells were cytospun, resuspended in RPMI medium and counted in a hemocytometer. For phagocytosis assays, the cells were allowed to adhere to coverslips for 1 h at 37³C and the monolayers formed were rinsed in RPMI medium and incubated with the bacteria (100 bacteria per phagocyte). Cell viability was estimated by trypan blue exclusion [22].
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2.3. Apoptosis assay Microscopic detection of apoptosis [23] was carried out both by identi¢cation of cells bearing condensed and fragmented nuclei in HE-stained preparations and by an in situ terminal deoxynucleotidyl transferase nick end labelling assay (TUNEL). For both assays, monolayers of phagocytic cells were incubated with bacteria for 30 min in RPMI medium at 37³C, the monolayers were then washed and incubated for 15 min with gentamicin (100Wg ml31 in RPMI medium) to remove non-ingested bacteria [24] and re-incubated in RPMI to complete 4 h of incubation and ¢nally ¢xed in absolute methanol. The TUNEL assay was carried out as follows. The cell monolayers were incubated at 37³C for 1 h in a medium containing 0.02 M 5-bromo-2P-deoxyuridine (BrdU), 30 U of terminal deoxynucleotidyl transferase (TdT), 0.1 M sodium cacodylate, 2 mM CoCl2 , 50 Wg ml31 BSA, 30 mM Tris-HCl and 4 mM of each dCTP, dGTP and dATP (Sigma). After the hybridization incubation, the preparations were rinsed with 50% formamide, then rinsed three times with 0.3 M NaCl containing 0.03 M sodium citrate, followed by three rinses in phosphate-bu¡ered saline (PBS) containing 0.05% Nonidet P-40 (NP bu¡er). Mouse anti-BrdU antibody (Sigma) diluted 1:200 in PBS was then applied over the cell monolayers for 30 min, the preparations were rinsed three times in NP bu¡er and incubated for 30 min with anti-mouse secondary antibody conjugated with FITC (Sigma). The preparations were analyzed in a £uorescence microscope (Zeiss) using a FITC ¢lter cube, background labelling was assessed on duplicate preparations incubated without TdT. 2.4. Analysis of DNA fragmentation DNA was extracted by the phenol-chloroform method [25] and fractionated in a 1% agarose gel in Tris-borate-EDTA bu¡er. Brie£y, 106 peritoneal phagocytic cells were co-incubated with 108 bacteria for 30 min, 100 Wg ml31 of gentamicin was added to kill extracellular bacteria and after 10 min, the cells were pelleted and resuspended in RPMI medium and re-incubated to complete 4 h. Control preparations were similarly processed, but with no addition of the bacteria. The cells were then pelleted, treated with
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the lysis bu¡er (10 mM Tris-HCl, 10 mM EDTA, 0.5% sodium dodecyl sulfate) and incubated at 56³C for 2 h. This preparation was treated with 100 Wg of RNAse A for 30 min at 37³C and the DNA was extracted with phenol-chloroform, precipitated overnight at 320³C in 5 M NaCl and pelleted with ethanol. The pellet was resuspended in 30 Wl of Tris-EDTA bu¡er and fractionated in a 1% agarose gel. The DNA was stained with ethidium bromide and visualized and photographed on an Image Master (Pharmacia).
3. Results 3.1. E. coli (UEL 17) induces macrophage apoptosis in vitro Macrophages infected with E. coli (UEL 17) presented the nuclear condensation and fragmentation pattern characteristic of apoptosis (Fig. 1B). Samples of the same macrophage population incubated with no bacteria presented normal morphology (Fig. 1A). DNA extracted from phagocytes incubated with E. coli showed a ladder pattern characteristic of apoptosis on agarose gel electrophoresis (Fig. 2). Fluorescence of the nuclei of infected cells, which reacted with BrdU on TUNEL assays, shows apoptotic cells (Fig. 3B), whereas the nuclei of non-infected cells did not react with the £uorescent FITC-conjugated antibody (Fig. 3A). Macrophages infected with S. £exneri, used as a positive control of apoptosis, showed a £uorescence pattern (Fig. 3D) similar to that preTable 1 E¡ect of cytochalasin D on macrophage apoptosis induced by E. coli Condition
No bacteria added E. coli S. £exneri
Apoptotic macrophages (%) Control
Cytochalasin D
3.5 þ 1.9 55.6 þ 1.4 42.5 þ 2.1
3.2 þ 1.9 13.1 þ 9.5 15.5 þ 2.0
The percentage of macrophage apoptosis was determined 4 h after infection with the bacteria, v 100 HE-stained macrophages were scored for the presence of apoptosis. S. £exneri was used as a positive control of apoptosis. Values are means þ S.D. of at least three independent experiments. P 6 0.05 for infected macrophages untreated versus treated with cytochalasin D.
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Fig. 1. Morphology of murine peritoneal macrophages infected with E. coli (UEL 17). Macrophages were either incubated with no bacteria (A) or incubated with viable (B) or heat-inactivated E. coli (C). Arrows on C point to examples of macrophages containing bacteria. After 4 h of incubation, the cells were stained with HE (100 bacteria per cell, magni¢cation U1000).
sented by the cells infected with E. coli. About 55% of macrophages were apoptotic 4 h after infection with E. coli (Table 1).
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3.2. Heat-inactivated E. coli does not induce macrophage apoptosis Macrophages co-incubated with heat-inactivated E. coli did not show the nuclear condensation and fragmentation characteristic of apoptosis (Fig. 1C). DNA extracted from cells incubated with heat-inactivated bacteria did not show the ladder pattern of fragmentation on agarose gel electrophoresis, remaining on the top of the gel (electrophoretogram not shown). That the heat-inactivated bacteria were phagocytosed is suggested by the presence of many bacteria surrounded by a clear halo within the contours of the macrophages (Fig. 1C). Similar preparations stained with acridine orange show, under the UV microscope, that many red (dead) bacteria are clustered inside the macrophages and segregated outside the nuclear region (UV micrograph not shown). 3.3. Inhibition of phagocytosis by cytochalasin D abolishes apoptosis induced by E. coli Macrophages pre-incubated with cytochalasin D (2 Wg ml31 ) for 30 min and then infected with E. coli did not react with the £uorescent tag on TUNEL assays (Fig. 3C). On optical microscopy of HEstained preparations, the nuclei of macrophages pretreated with cytochalasin D and infected with E. coli were not condensed or fragmented (micrograph not shown), the cell morphology being indistinguishable from that of normal macrophages shown on Fig. 1A. Pretreatment of macrophages with cytochalasin D reduced the percentage of apoptotic cells (Table 1) from 55.6 þ 1.4 to 13.1 þ 9.5 (S.D.). 3.4. Both the colicin V-producing strain E. coli UEL 17 and an isogenic Col V3 derivative induce macrophage apoptosis The wild-type E. coli UEL 17 used in the experiments described above carries three large plasmids, one of which encodes colicin V. An isogenic strain which lacks this plasmid (E. coli UEL 17 Col V3 ) was also capable of inducing apoptosis, as indicated by analysis of DNA fragmentation (Fig. 2). Macrophages infected with the Col V3 strain for 30 min and then re-incubated to complete 4 h of incubation presented nuclear fragmentation and condensation
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Fig. 2. DNA fragmentation of murine peritoneal macrophages infected with E. coli (UEL 17). Cells were co-incubated with bacteria for 30 min, treated with gentamicin (100 Wg ml31 ) and cytospun and re-incubated (4 h total). Lanes: (A) molecular mass markers; (B, C) controls, non-infected; (D, E) cells infected with E. coli Col V ; (F, G) cells infected with isogenic E. coli Col V3 .
(micrograph not shown) similar to that caused by the Col V strain shown on Fig. 1B.
4. Discussion Avian pathogenic strains of E. coli can cause upper respiratory infections that spread to the lower respiratory tract and subsequently develop into colisepticemia in poultry. The mechanisms and virulence factors employed by those bacteria to circumvent the host defences and cross through the epithelial barriers are not completely known. In this study, we observed that a strain of avian E. coli caused macrophage apoptosis in vitro, which suggests that induction of apoptosis might favor the invasiveness of those bacteria. That the mechanism of macrophage death was predominantly apoptosis was shown by the identi¢cation of nuclear fragmentation and condensation by optical microscopy, by in situ analysis of DNA fragmentation by TUNEL assays and by the presence of the characteristic ladder pattern of DNA-banding on agarose gel elec-
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Fig. 3. TUNEL reaction in infected murine peritoneal macrophages. Cells were incubated with E. coli for 30 min, washed with gentamicin (100 Wg ml31 ) to remove non-ingested bacteria and re-incubated to complete 4 h of incubation. TUNEL reaction was used to label 3P-OH ends with £uorescein. Cells were either non-infected (A) or infected (B) or pretreated with cytochalasin D for 30 min and then infected (C). Macrophages infected with S. £exneri M90T were used as a positive control of apoptosis (D). Bars represent 10 Wm.
trophoresis. The time-course of apoptosis induction by E. coli was similar to that reported for S. £exneri [14], Salmonella typhimurium [17] and Yersinia enterocolitica [18]. 1^2 h after co-incubation of E. coli with macrophages, apoptosis could already be detected (data not shown) and after 4 h of incubation, about 55% of macrophages were apoptotic. This relatively short time-course of apoptosis induction seems to be common to phagocytosed bacteria and to invasive bacteria, in contrast to the longer times taken for the induction of apoptosis by bacteria adapted to intracellular living, such as Mycobacterium tuberculosis [26]. Inhibition of phagocytosis by cytochalasin D prevented the induction of apoptosis by E. coli, indicating that the bacteria must be intracellularly located to induce apoptosis. Similar results were observed
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with S. £exneri [14] and S. typhimurium [17], whose ability to induce macrophage apoptosis is blocked by cytochalasin D. Moreover, heat-inactivated E. coli were phagocytosed but failed to induce apoptosis. Therefore, these results show that the induction of apoptosis by E. coli depends both on the bacteria being intracellularly located and viable. This requirement for bacterial viability suggests that some protein(s) secreted by E. coli mediate the induction of apoptosis, as has been observed for S. £exneri [19,21] and for Salmonella spp. [20]. Colicin V-producing strains of E. coli seem to be more virulent than non-producers [6,7]. A direct role of colicin V as a virulence factor has not been conclusively demonstrated, although it is known that colicins disrupt the membrane potential of target cells [8^10]. Because the insertion of colicins on their
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target membranes is known to depend on the potential di¡erence across those membranes and given that the phagolysosome is at a pH di¡erent from that of the cytoplasm, we hypothesized that colicin V secreted by phagocytosed E. coli could be inserted in the membrane of the phagosome and activate apoptosis. We found that both the colicin V-producing wild-type strain of E. coli and one isogenic Col V3 strain induced macrophage apoptosis, showing that the production of colicin V by E. coli is not necessary for its ability to induce apoptosis. The results of this study suggest, therefore, that the induction of macrophage apoptosis by avian septicemic E. coli might be a pathogenic mechanism employed by those bacteria to circumvent the host cellular defences and that to induce apoptosis, the bacteria must be both intracellularly located and viable. Moreover, these results also show that the production of colicin V by E. coli is not required for its ability to induce macrophage apoptosis.
[10]
[11]
[12]
[13]
[14]
[15]
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[17]
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