Bacteroides fragilis Supernatant Extracts Enriched in Phenylacetic Acid Induce a Cytotoxic Effect in Mammalian Cells

Laís S. Falcão^1,2, Eduardo N. F. Antunes², Eliane O. Ferreira², Heidi Pauer², Maria Teresa V. Romanos², Rossiane C. Vommaro³, Sérgio H. Seabra^3,4, Daniela S. Alviano², Celuta S. Alviano², Antonio Jorge R. da Silva⁵, Leandro A. Lobo^2*, Regina Maria C. P. Domingues²

¹Departamento de Ciências Básicas, Pólo Universitário de Nova Friburgo, Universidade Federal Fluminense, Nova Friburgo, Brasil.
²Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil.
³Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil.
⁴Centro Universitário Estadual da Zona Oeste—UEZO, Rio de Janeiro, Brasil.
⁵Instituto de Pesquisa Produtos Naturais, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil.

DOI: 10.4236/aim.2015.510077                

Abstract

Bacteroides species are nearly half of the fecal flora community and some are host symbionts crucial to host nutrition and systemic immunity. Among Bacteroides species B. fragilis strains are considered to be the opportunistic ones, being the most isolated anaerobic bacteria in clinical samples. Cell-free supernatants of 65 B. fragilis strains were assayed and they were capable of inducing vacuolating phenotype on Vero cells lineage. The supernatant of the Bacteroides fragilis ATCC 23745 strain was elicited to have the strongest vacuolating effect on Vero cells monolayers and peritoneal macrophages. Some drastic cell alterations were observed, such as a general disorganization of cytoplasm and chromatin condensation, evidencing cell death. By transmission electron microscopy it was confirmed that the vacuoles observed were, in fact, swollen mitochondria. An immunocytochemical assay, TUNEL, was used to confirm this hypothesis and showed that Vero cells and peritoneal macrophages were dying by apoptotic process after exposition of B. fragilis cell-free supernatant. Physical analysis of the apoptotic factor has revealed properties similar to short-chain fatty acids. After gas chromatography and mass spectrometry analysis, phenylacetic acid (PA) was characterized as the major compound present in the most purified active fraction. We believe that the PA is responsible for the pro-apoptotic effect elicited by the supernatant of B. fragilis cultures.

Keywords

Bacteroides fragilis, Vacuolization, Apoptosis, Vero cells Lineage, Peritoneal Macrophages, Phenylacetic Acid

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	DiBaise, J.K., Zhang, H., Crowell, M.D., et al. (2008) Gut Microbiota and Its Possible Relationship with Obesity. Mayo Clinic Proceedings, 83, 460-469. http://dx.doi.org/10.4065/83.4.460
[2]	Falk, P.G., Hooper, L.V., Midtvedt, T. and Gordon, J.I. (1998) Creating and Maintaining the Gastrointestinal Ecosystem: What We Know and Need to Know from Gnotobiology. Microbiology and Molecular Biology Reviews, 62, 1157-1170.
[3]	Wexler, H.M. (2007) Bacteroides: The Good, the Bad, and the Nitty-Gritty. Clinical Microbiology Reviews, 20, 593-621. http://dx.doi.org/10.1128/CMR.00008-07
[4]	Sears, C.L. (2009) Enterotoxigenic Bacteroides Fragilis: A Rogue among Symbiotes. Clinical Microbiology Reviews, 22, 349-369. http://dx.doi.org/10.1128/CMR.00053-08
[5]	Mayhew, J.W., Onderdonka, B. and Gorbach, S.L. (1975) Effects of Time and Growth Media on Short-Chain Fatty Acid Production by Bacteroides Fragilis. Applied Microbiology, 29, 472-475.
[6]	Ginsburg, E., Salomon, D., Sreevalsan, T. and Freese, E. (1973) Growth Inhibition and Morphological Changes Caused by Lipophilic Acids in Mammalian Cells. Proceedings of the National Academy of Sciences of USA, 70, 2457-2461. http://dx.doi.org/10.1073/pnas.70.8.2457
[7]	Touw, J.J., van Steenbergen, T.J. and De Graaff, J. (1982) Butyrate: A Cytotoxin for Vero Cells Produced by Bacteroides gingivalis and Bacteroides asaccharolyticus. Antonie van Leeuwenhoek, 48, 315-325. http://dx.doi.org/10.1007/BF00418285
[8]	Grenier, D. and Mayrand, D. (1985) Cytotoxic Effects of Culture Supernatants of Oral Bacteria and Various Organic Acids on Vero Cells. Canadian Journal of Microbiology, 31, 302-304. http://dx.doi.org/10.1139/m85-057
[9]	Ohkusa, T., Okayasu, I., Ogihara, T., et al. (2003) Induction of Experimental Ulcerative Colitis by Fusobacterium varium Isolated from Colonic Mucosa of Patients with Ulcerative Colitis. Gut, 52, 79-83. http://dx.doi.org/10.1136/gut.52.1.79
[10]	Antunes, E.N.F., Ferreira, E.O., Falcao, L.S., et al. (2004) Non-Toxigenic Pattern II and III Bacteroides Fragilis Strains: Coexistence in the Same Host. Research in Microbiology, 155, 522-524. http://dx.doi.org/10.1016/j.resmic.2004.04.008
[11]	Rocha, E.R., Owens, G., Smith, C.J. and Carolina, N. (2000) The Redox-Sensitive Transcriptional Activator OxyR Regulates the Peroxide Response Regulon in the Obligate Anaerobe Bacteroides fragilis. Journal of Bacteriology, 182, 5059-5069. http://dx.doi.org/10.1128/jb.182.18.5059-5069.2000
[12]	Vieira, J.M.B.D., Vallim, D.C., Ferreira, E.O., et al. (2005) Bacteroides fragilis Interferes with iNOS Activity and Leads to Pore Formation in Macrophage Surface. Biochemical and Biophysical Research Communications, 326, 607-613. http://dx.doi.org/10.1016/j.bbrc.2004.11.085
[13]	Favre-Bonte, S., Darfeuille-Michaud, A. and Forestier, C. (1995) Aggregative Adherence of Klebsiella pneumoniae to Human Intestine-407 Cells. Infection and Immunity, 63, 1318-1328.
[14]	Matsumoto, T., Hayasaki, T., Nishimura, Y., et al. (2006) Butyrate Induces Necrotic Cell Death in Murine Colonic Epithelial Cell MCE301. Biological and Pharmaceutical Bulletin, 29, 2041-2045. http://dx.doi.org/10.1248/bpb.29.2041
[15]	Apostolova, N., Blas-Garcia, A. and Esplugues, J.V. (2011) Mitochondria Sentencing about Cellular Life and Death: A Matter of Oxidative Stress. Current Pharmaceutical Design, 17, 4047-4060. http://dx.doi.org/10.2174/138161211798764924
[16]	Zimmermann, K.C. and Green, D.R. (2001) How Cells Die: Apoptosis Pathways. Journal of Allergy and Clinical Immunology, 108, S99-S103. http://dx.doi.org/10.1067/mai.2001.117819
[17]	Leunk, R.D. (1991) Production of a Cytotoxin by Helicobacter pylori. Reviews of Infectious Diseases, 13, S686-S689. http://dx.doi.org/10.1093/clinids/13.supplement_8.s686
[18]	Sakurai, N., Koike, K.A., Irie, Y. and Hayashi, H. (1994) The Rice Culture Filtrate of Bacillus cereus Isolated from Emetic-Type Food Poisoning Causes Mitochondrial Swelling in a HEp-2 Cell. Microbiology and Immunology, 38, 337-343. http://dx.doi.org/10.1111/j.1348-0421.1994.tb01788.x
[19]	Figueroa-Arredondo, P., Heuser, J.E., Akopyants, N.S., et al. (2001) Cell Vacuolation Caused by Vibrio cholerae Hemolysin. Infection and Immunity, 69, 1613-1624. http://dx.doi.org/10.1128/IAI.69.3.1613-1624.2001
[20]	Sakurazawa, T. and Ohkusa, T. (2005) Cytotoxicity of Organic Acids Produced by Anaerobic Intestinal Bacteria on Cultured Epithelial Cells. Journal of Gastroenterology, 40, 600-609. http://dx.doi.org/10.1007/s00535-005-1594-z
[21]	Ariake, K., Ohkusa, T., Sakurazawa, T., et al. (2000) Roles of Mucosal Bacteria and Succinic Acid in Colitis Caused by Dextran Sulfate Sodium in Mice. Journal of Medical and Dental Sciences, 47, 233-241.
[22]	Schmidt, S., Westhoff, T.H., Krauser, P., et al. (2008) The Uraemic Toxin Phenylacetic Acid Impairs Macrophage Function. Nephrology Dialysis Transplantation, 23, 3485-3493. http://dx.doi.org/10.1093/ndt/gfn266
[23]	Kim, Y., Cho, J.-Y., Kuk, J.-H., et al. (2004) Identification and Antimicrobial Activity of Phenylacetic Acid Produced by Bacillus licheniformis Isolated from Fermented Soybean, Chungkook-Jang. Current Microbiology, 48, 312-317. http://dx.doi.org/10.1007/s00284-003-4193-3
[24]	Kampa, M., Alexaki, V.-I., Notas, G., et al. (2004) Antiproliferative and Apoptotic Effects of Selective Phenolic Acids on T47D human Breast Cancer Cells: Potential Mechanisms of Action. Breast Cancer Research, 6, R63-R74. http://dx.doi.org/10.1186/bcr752
[25]	Van Assche, P.F. (1978) Differentiation of Bacteroides fragilis Species by Gas Chromatographic Detection of Phenylacetic Acid. Journal of Clinical Microbiology, 8, 614-615.