Microcin E492, a channel-forming bacteriocin from Klebsiella pneumoniae, induces apoptosis in some human cell lines Claudio Hetz*†, Mari´a Rosa Bono*, Luis Felipe Barros†‡, and Rosalba Lagos*§ *Departamento de Biologı´a,Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile; †Instituto de Ciencias Biome´dicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago, Chile; and ‡Centro de Estudios Cientı´ficos,Arturo Prat 514, Valdivia, Chile Communicated by Jorge E. Allende, University of Chile, Santiago, Chile, December 31, 2001 (received for review August 20, 2001) The cytotoxic effect of microcin E492, a low-molecular-mass chan- Apoptosis is a genetically determined form of cell death that nel-forming bacteriocin (7,887 Da) produced by a strain of Kleb- plays a central role during development and homeostasis of siella pneumoniae, was characterized in HeLa cells. At low (5 multicellular organisms (13, 14). Necrotic cell death is usually the ␮g͞ml) and intermediate (10 ␮g͞ml) concentrations, microcin E492 consequence of physical injury and does not involve the active induced biochemical and morphological changes typical of apo- participation of the cell. Apoptosis can be distinguished from ptosis, such as cell shrinkage, DNA fragmentation, extracellular necrosis on the basis of several morphological as well as bio- exposure of phosphatidylserine, caspase activation, and loss of chemical parameters, such as nuclear condensation, loss of cell mitochondrial membrane potential. Treatment with zVAD-fmk, a volume (shrinkage), DNA fragmentation (14), and phosphati- general caspase inhibitor, completely blocked the cytotoxic effect dylserine exposure to the outer face of plasma membrane (15). ␮ ͞ of this bacteriocin. At higher microcin concentrations (>20 g ml) This kind of cell death avoids spillage of intracellular contents in a necrotic phenotype was observed. Induction of apoptosis by contrast to necrotic cell death, typified by cell and organelle microcin E492 was associated with the release of calcium from swelling and membrane disruption, resulting in an inflammatory intracellular stores, probably after microcin-triggered ion channel response (15). The central executioners of apoptosis are a set of formation. Microcin E492 also presented a cytotoxic effect on cysteine proteases that are part of a large protein family known Jurkat and RJ2.25 cells, but had no effect on KG-1 cells nor on a as caspases (16). primary culture of human tonsil endothelial cells, suggesting that Some bacterial toxins share common features with bacterio- there is a specific interaction of the bacteriocin with components cins, such as insertion into or transport across the plasma of the target cell surface. This report describes a bacteriocin that membrane, recognition of the target cell’s membrane, pore has the capacity to induce apoptosis in human cell lines. formation, etc. The known action of bacterial toxins and bacte- riocins has been restricted to eukaryotic cells and bacteria, acteriocins are antimicrobial proteins produced by bacteria respectively, probably because of specific interactions between Bthat are active against closely related species. They are the receptor and the toxin. The only bacteriocin reported to be produced by almost all major lineages of Eubacteria and in lytic for eukaryotic as well as prokaryotic cells is a cytolysin from Archaebacteria (1) and secreted to the extracellular medium Enterococcus faecalis (17). Although it is known that this toxin where they recognize specific receptors on the surface of sen- induces cell lysis, no evidence has been presented as to whether sitive bacterial cells (2). It has been proposed that the primary cytotoxicity is caused by apoptosis or necrosis. role of bacteriocins is to mediate population dynamics within The aim of this work was to study the ability of microcin E492 closely related species (1). Bacteriocins can induce toxicity by a to induce apoptosis. Microcin E492 is a low-molecular-mass variety of mechanisms, most notably the formation of membrane (7,887 Da) channel-forming bacteriocin (18, 19) produced by pores. These compounds have received increasing attention for Klebsiella pneumoniae RYC492, which has a rather broad target their potential use as preservatives in the food industry and as range, being active on strains of the family Enterobacteriaceae antibiotics for clinical usage (3–6). (20). The genetic determinants involved in the production of Bacteria also produce other toxic proteins, released by patho- gens into the host’s body, that are important virulence factors active microcin and its immunity protein have been cloned and causing disease symptoms (tetanus, diphtheria, cholera, etc.). expressed in Escherichia coli (21) and comprise a 13-kb DNA These toxic agents are commonly called bacterial toxins and may fragment encoding for at least 10 genes, necessary for the attack a variety of different cell types (cytotoxins) or specific cell synthesis and export of active microcin (22). Analysis of the types (neurotoxins, hemolysins, etc). Among these bacterial amino acid sequence of microcin E492 indicates that this bac- toxins some can also form membrane pores and kill eukaryotic teriocin does not share structural motif with any other known cells by disrupting the membrane permeability barrier. Lately, protein, and thus, it seems that this microcin belongs to a novel increasing interest has developed in the study of some toxins for class of pore-forming bacteriocins not related to colicin-like their potential therapeutic use as antitumoral drugs. Thus, channel-forming bacteriocin from Gram-negative bacteria or to cytotoxic studies of toxins such as verotoxin 1 (7) and a derivative channel-forming bacteriocins from Gram-positive bacteria (19). of a Pseudomonas exotoxin (8) have demonstrated that these Results presented in this article show that in certain human cell toxins display antitumor activity in vitro and in vivo in ovarian and lines microcin E492 promotes morphological and biochemical breast cancer, respectively. Another aspect that has aroused changes typical of apoptosis. considerable interest is the role of toxin-producing pathogens in promoting apoptosis (9). In contrast to necrosis, apoptosis Abbreviations: AU, antibiotic units; PI, propidium iodide; DiOC6, dihexyloxacarbocyanine; eliminates cells without inducing an inflammatory response, i.e., LDH, lactate dehydrogenase; LFA-1, lymphocyte function-associated antigen 1; TNF-␣, in an immunologically silent manner subverting the normal host tumor necrosis factor ␣. defense responses. Among the pore-forming toxins that induce § ␣ To whom reprint requests should be addressed. E-mail: [email protected]. apoptosis is -toxin, the major cytolysin from Staphylococcus The publication costs of this article were defrayed in part by page charge payment. This aureus (10), aerolysin, produced by Aeromonas hydrophyla (11), article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. and the porin PorB from Neisseria gonorrhoeae (12). §1734 solely to indicate this fact. 2696–2701 ͉ PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 www.pnas.org͞cgi͞doi͞10.1073͞pnas.052709699 Downloaded by guest on September 26, 2021 Table 1. Effect of microcin E492 on different human cells types Cell type Cell survival, % Jurkat 4 Ϯ 3 HeLa 56 Ϯ 6 RJ2.2.5 57 Ϯ 11 Ramos 79 Ϯ 19 KG-1 91 Ϯ 1 AMG-3 99 Ϯ 1 Cells were incubated for 24 h in the absence (untreated control) or presence of 680 AU/ml of microcin E492. The percentages of cell survival were deter- mined in respect to the untreated control and are the averages of three independent measurements. Cells were stained with PI, and cell death was determined by flow cytometry analysis. Materials and Methods Isolation, Purification, and Fluorescent Labeling of Microcin E492. Microcin E492 was extracted and purified (20) from the super- natant cultures of E. coli VCS257pJEM15. Microcin activity was estimated and expressed in arbitrary antibiotic units (AU) (23). Normally, 10 ␮g of microcin E492 corresponded to 500 AU. Labeling of microcin with fluorescamine and PAGE were car- ried out as described (21). Cell Lines. The following human cell lines (American Type Culture Collection) were used: HeLa, an epithelial cell line derived from human cervix carcinoma; KG-1, a monocyte- macrophage cell line; RJ2.25, a variant of the Raji B-LCL (24); Jurkat, a T cell derived from acute T cell leukemia, and Ramos, a B cell line originated from Burkitt’s lymphoma. The human endothelial cells from human tonsils (AMG-3) were isolated as described (25). Ramos, Jurkat, RJ2.2.5, KG-1, and AMG-3 cells were maintained in RPMI 1640 medium supplemented with 10% BIOCHEMISTRY FCS and antibiotics. HeLa cell line were maintained in DMEM with 10% FCS and antibiotics. Cytotoxicity and Apoptosis Assays. Cells were seeded at 2 ϫ 104 cells͞well in 96-well plates 24 h before microcin E492 addition, further incubated for the indicated time with microcin E492, ␮ ͞ Fig. 1. Microcin E492 induces cell death with DNA fragmentation in a trypsinized and washed, suspended in PBS, stained with 1 g ml dose-dependent manner. HeLa cells (0.4 ϫ 106 cells in 1 ml) were incubated propidium iodide (PI), and analyzed by using a FACScan during 24 h under different conditions and analyzed by the PI exclusion (Becton Dickinson) with CELL QUEST software. The loss of method. (A) Untreated control. NT, nontreated. (B) Cells incubated with 256 mitochondrial membrane potential was assessed by using 3,3Ј- AU microcin E492. (C) Cells incubated with 1,024 AU microcin E492. Dead cells dihexyloxacarbocyanine [DiOC (3)] (Molecular Probes) as de- permeable to PI are indicated in A–C as M1 and M2 populations. M0 popula- 6 tion represents viable cells, not permeable to PI. mcc, microcin E492. (D) Cells scribed (26). Exposure of phosphatidylserine to the outer leaflet incubated with a preparation from E. coli VCS257np108 with an OD280 equiv- of the plasma membrane was determined by flow cytometry with alent to 10 ␮g. (Inset) A PAGE of microcin E492 (labeled with fluorescamine) annexin V coupled to FITC (Annexin-V-FLUOS staining kit, purified from the microcin-producing strain E.