Abstract
We have previously demonstrated that Yersinia ruckeri resists cecropin B in an inducible manner. In this study, we sought to identify the molecular changes responsible for the inducible cecropin B resistance of Y. ruckeri. Differences in gene expression associated with the inducible resistance were investigated. Cultures of Y. ruckeri were exposed to a sublethal concentration of cecropin B and resultant changes in the messenger RNA population of the bacteria were assayed using the differential display reverse transcription polymerase chain reaction (DD-RT-PCR). A single band was consistently increased in intensity in all repeats of the experiment. The band was excised, cloned, sequenced, and used to screen a Y. ruckeri genomic DNA library. The DD-RT-PCR fragment shared 100% identity to the cDNA sequence of an ATP-dependent endonuclease of the overcome lysogenization defect (OLD) family of Y. ruckeri 29473. The genomic clone that was recovered was not identical to the DD-RT-PCR clone, but harbored a gene for a secreted endonuclease 1 (nucM) homologue. It was determined that transcription of the gene was upregulated following exposure to cecropin B via RT-PCR. Furthermore, an increase in the nuclease activity of culture supernatants of Y. ruckeri following exposure to cecropin B was demonstrated. These findings demonstrate that cecropin B exposure increases the expression of at least two endonucleases in Y. ruckeri. The production and secretion of an endonuclease by Y. ruckeri in response to an antimicrobial peptide indicates the involvement of both intracellular and extracellular DNA in the toxic effects of cecropin B.
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Agerberth B, Charo J, Werr J, Olsson B, Idali F, Lindbom L, Kiessling R, Jornvall H, Wigzell H, Gudmundsson GH (2000) The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations. Blood 96:3086–3093
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Bowdish DM, Davidson DJ, Hancock RE (2005) A re-evaluation of the role of host defence peptides in mammalian immunity. Current Protein and Peptide Science 6:35–51
Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A (2004) Neutrophil extracellular traps kill bacteria. Science 303:1532–1535
Cole AM, Weis P, Diamond G (1997) Isolation and characterization of pleurocidin, an antimicrobial peptide in the skin secretions of winter flounder. J Biol Chem 272:12008–12013
Ernst RK, Guina T, Miller SI (1999) How intracellular bacteria survive: surface modifications that promote resistance to host innate immune responses. J Infect Dis 179(Suppl 2):S326–S330
Fleming JT, Yao WH, Sayler GS (1998) Optimization of differential display of prokaryotic mRNA: application to pure culture and soil microcosms. Appl Environ Microbiol 64:3698–3706
Gibello A, Blanco MM, Moreno MA, Cutuli MT, Domenech A, Dominguez L, JF FG (1999) Development of a PCR assay for detection of Yersinia ruckeri in tissues of inoculated and naturally infected trout. Appl Environ Microbiol 65:346–350
Groisman EA, Parra-Lopez C, Salcedo M, Lipps CJ, Heffron F (1992) Resistance to host antimicrobial peptides is necessary for Salmonella virulence. Proc Natl Acad Sci U S A 89:11939–11943
Guo L, Lim KB, Poduje CM, Daniel M, Gunn JS, Hackett M, Miller SI (1998) Lipid A acylation and bacterial resistance against vertebrate antimicrobial peptides. Cell 95:189–198
Jacobsen F, Baraniskin A, Mertens J, Mittler D, Mohammadi-Tabrisi A, Schubert S, Soltau M, Lehnhardt M, Behnke B, Gatermann S, Steinau HU, Steinstraesser L (2005) Activity of histone H1.2 in infected burn wounds. J Antimicrob Chemother 55:735–741
Kawasaki H, Iwamuro S (2008) Potential roles of histones in host defense as antimicrobial agents. Infect Disord Drug Targets 8:195–205
Kristian SA, Datta V, Weidenmaier C, Kansal R, Fedtke I, Peschel A, Gallo RL, Nizet V (2005) D-alanylation of teichoic acids promotes group a streptococcus antimicrobial peptide resistance, neutrophil survival, and epithelial cell invasion. J Bacteriol 187:6719–6725
Miele G, MacRae L, McBride D, Manson J, Clinton M (1998) Elimination of false positives generated through PCR re-amplification of differential display cDNA. Biotechniques 25:138–144
Parseghian MH, Luhrs KA (2006) Beyond the walls of the nucleus: the role of histones in cellular signaling and innate immunity. Biochem Cell Biol 84:589–604
Patrzykat A, Friedrich CL, Zhang L, Mendoza V, Hancock RE (2002) Sublethal concentrations of pleurocidin-derived antimicrobial peptides inhibit macromolecular synthesis in Escherichia coli. Antimicrob Agents Chemother 46:605–614
Prithivirajsingh S, Mishra SK, Mahadevan A (2001) Functional analysis of a chromosomal arsenic resistance operon in Pseudomonas fluorescens strain MSP3. Mol Biol Rep 28:63–72
Richards RC, O'Neil DB, Thibault P, Ewart KV (2001) Histone H1: an antimicrobial protein of Atlantic salmon (Salmo salar). Biochem Biophys Res Commun 284:549–555
Sallum UW, Chen TT (2008) Inducible resistance of fish bacterial pathogens to the antimicrobial peptide cecropin B. Antimicrob Agents Chemother 52:3006–3012
Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York
Sarmasik A, Warr G, Chen TT (2002) Production of transgenic medaka with increased resistance to bacterial pathogens. Mar Biotechnol (NY) 4:310–322
Sumby P, Barbian KD, Gardner DJ, Whitney AR, Welty DM, Long RD, Bailey JR, Parnell MJ, Hoe NP, Adams GG, Deleo FR, Musser JM (2005) Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response. Proc Natl Acad Sci U S A 102:1679–1684
Tobback E, Decostere A, Hermans K, Haesebrouck F, Chiers K (2007) Yersinia ruckeri infections in salmonid fish. J Fish Dis 30:257–268
Tollin M, Bergman P, Svenberg T, Jornvall H, Gudmundsson GH, Agerberth B (2003) Antimicrobial peptides in the first line defence of human colon mucosa. Peptides 24:523–530
Tzeng YL, Ambrose KD, Zughaier S, Zhou X, Miller YK, Shafer WM, Stephens DS (2005) Cationic antimicrobial peptide resistance in Neisseria meningitidis. J Bacteriol 187:5387–5396
von Kockritz-Blickwede M, Goldmann O, Thulin P, Heinemann K, Norrby-Teglund A, Rohde M, Medina E (2008) Phagocytosis-independent antimicrobial activity of mast cells by means of extracellular trap formation. Blood 111:3070–3080
von Kockritz-Blickwede M, Nizet V (2009) Innate immunity turned inside-out: antimicrobial defense by phagocyte extracellular traps. J Mol Med 87:775–783
Wang Y, Xu Z, Jia A, Chen J, Mo Z, Zhang X (2009) Genetic diversity between two Vibrio anguillarum strains exhibiting different virulence by suppression subtractive hybridization. Wei Sheng Wu Xue Bao 49:363–371
Yeaman MR, Yount NY (2007) Unifying themes in host defence effector polypeptides. Nat Rev Microbiol 5:727–740
Acknowledgements
We would like to acknowledge Professor Joerg Graf of the Department of Molecular and Cell Biology at the University of Connecticut, Storrs, for his guidance in the construction and screening of the Y. ruckeri genomic library. This work was supported by a grant from United States Department of Agriculture (CONTR #58-1930-0-009) to T.T.C.
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Sallum, U.W., Chen, T.T. Molecular Cloning of Cecropin B Responsive Endonucleases in Yersinia ruckeri . Mar Biotechnol 13, 56–65 (2011). https://doi.org/10.1007/s10126-010-9269-z
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DOI: https://doi.org/10.1007/s10126-010-9269-z