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Research article

Clinical Microbiology and Biomedical Sciences

Role of LuxIR Homologue AnoIR in Acinetobacter nosocomialis and the Effect of Virstatin on the Expression of anoR Gene

Man Hwan Oh 1 and Chul Hee Choi 2*

1Department of Nanobiomedical Science, Dankook University, Cheonan 330-714, Republic of Korea, 2Department of Microbiology and Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Daejeon 301-747, Republic of Korea

Received: April 27, 2015; Accepted: May 14, 2015

J. Microbiol. Biotechnol. 2015; 25(8): 1390-1400

Published August 28, 2015 https://doi.org/10.4014/jmb.1504.04069

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

Quorum sensing is a process of cell-to-cell communication in which bacteria produce autoinducers as signaling molecules to sense cell density and coordinate gene expression. In the present study, a LuxI-type synthase, AnoI, and a LuxR-type regulator, AnoR, were identified in Acinetobacter nosocomialis, an important nosocomial pathogen, by sequence analysis of the bacterial genome. We found that N-(3-hydroxy-dodecanoyl)-L-homoserine lactone (OH-dDHL) is a quorum-sensing signal in A. nosocomialis. The anoI gene deletion was responsible for the impairment in the production of OH-dDHL. The expression of anoI was almost abolished in the anoR mutant. These results indicate that AnoI is essential for the production of OH-dDHL in A. nosocomialis, and its expression is positively regulated by AnoR. Moreover, the anoR mutant exhibited deficiency in biofilm formation. In particular, motility of the anoR mutant was consistently and significantly abolished compared with that of the wild type. The deficiency in the biofilm formation and motility of the anoR mutant was significantly restored by a functional anoR, indicating that AnoR plays important roles in the biofilm formation and motility. Furthermore, the present study showed that virstatin exerts its effects on the reduction of biofilm formation and motility by inhibiting the expression of anoR. Consequently, the combined results suggest that AnoIR is a quorum-sensing system that plays important roles in the biofilm formation and motility of A. nosocomialis, and virstatin is an inhibitor of the expression of anoR.

Keywords

Acinetobacter nosocomialis, quorum sensing, AnoIR, biofilm, motility, virstatin

References

  1. Bhargava N, Sharma P, Capalash N. 2010. Quorum sensing in Acinetobacter: an emerging pathogen. Crit. Rev. Microbiol. 36: 349-360.
    Pubmed CrossRef
  2. Callahan SM, Dunlap PV. 2000. LuxR- and acyl-homoserinelactonecontrolled non-lux genes define a quorum-sensing regulon in Vibrio fischeri. J. Bacteriol. 182: 2811-2822.
    Pubmed PMC CrossRef
  3. Chabane YN, M louka M B, A lexandre S, Nicol M, M arti S, Pestel-Caron M, et al. 2014. Virstatin inhibits biofilm formation and motility of Acinetobacter baumannii. BMC Microbiol. 14: 62.
    Pubmed PMC CrossRef
  4. Chow JY, Yang Y, Tay SB, Chua KL, Yew WS. 2014. Disruption of biofilm formation by the human pathogen Acinetobacter baumannii using engineered quorum-quenching lactonases. Antimicrob. Agents Chemother. 58: 1802-1805.
    Pubmed PMC CrossRef
  5. Clemmer KM, Bonomo RA, Rather PN. 2011. Genetic analysis of surface motility in Acinetobacter baumannii. Microbiology 157: 2534-2544.
    Pubmed PMC CrossRef
  6. Cook DM, Li P-L, Ruchaud F, Padden S, Farrand SK. 1997. Ti plasmid conjugation is independent of vir: reconstitution of the tra functions from pTiC58 as a binary system. J. Bacteriol. 179: 1291-1297.
    Pubmed PMC CrossRef
  7. Dijkshoorn L, Nemec A, Seifert H. 2007. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat. Rev. Microbiol. 5: 939-951.
    Pubmed CrossRef
  8. Gaddy JA, Tomaras AP, Actis LA. 2009. The Acinetobacter baumannii 19606 OmpA protein plays a role in biofilm formation on abiotic surfaces and in the interaction of this pathogen with eukaryotic cells. Infect. Immun. 77: 3150-3160.
    Pubmed PMC CrossRef
  9. Gray KM, Passador L, Iglewski BH, Greenberg EP. 1994. Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa. J. Bacteriol. 176: 3076-3080.
    Pubmed PMC CrossRef
  10. Hall-Stoodley L, Costerton JW, Stoodley P. 2004. Bacterial biofilms: from the natural environment to infectious diseases. Nat. Rev. Microbiol. 2: 95-108.
    Pubmed CrossRef
  11. Harshey RM. 2003. Bacterial motility on a surface: many ways to a common goal. Annu. Rev. Microbiol. 57: 249-273.
    Pubmed CrossRef
  12. Hung DT, Shakhnovich EA, Pierson E, Mekalanos JJ. 2005. Small-molecule inhibitor of Vibrio cholerae virulence and intestinal colonization. Science 310: 670-674.
    Pubmed CrossRef
  13. Hunger M, Schmucker R, Kishan V, Hillen W. 1990. Analysis and nucleotide sequence of an origin of DNA replication in Acinetobacter calcoaceticus and its use for Escherichia coli shuttle plasmids. Gene 87: 45-51.
    CrossRef
  14. Kim J, Park W. 2013. Identification and characterization of genes regulated by AqsR, a LuxR-type regulator in Acinetobacter oleivorans DR1. Appl. Microbiol. Biotechnol. 97: 6967-6978.
    Pubmed CrossRef
  15. Lee YC, Huang YT, Tan CK, Kuo YW, Liao CH, Lee PI, et al. 2011. Acinetobacter baumannii and Acinetobacter genospecies 13TU and 3 bacteraemia: comparison of clinical features, prognostic factors and outcomes. J. Antimicrob. Chemother. 66: 1839-1846.
    Pubmed CrossRef
  16. Nemec A, Krizova L, Maixnerova M, van der Reijden TJ, Deschaght P, Passet V, et al. 2011. Genotypic and phenotypic characterization of the Acinetobacter calcoaceticus–Acinetobacter baumannii complex with the proposal of Acinetobacter pittii sp. nov. (formerly Acinetobacter genomic species 3) and Acinetobacter nosocomialis sp. nov. (formerly Acinetobacter genomic species 13TU). Res. Microbiol. 162: 393-404.
    Pubmed CrossRef
  17. Niu C, Clemmer KM, Bonomo RA, Rather PN. 2008. Isolation and characterization of an autoinducer synthase from Acinetobacter baumannii. J. Bacteriol. 190: 3386-3392.
    Pubmed PMC CrossRef
  18. Oh MH, Lee JC, Kim J, Choi CH, Han K. 2015. Simple method for markerless gene deletion of multidrug-resistant Acinetobacter baumannii. Appl. Environ. Microbiol. 81: 3357-3368.
    Pubmed PMC CrossRef
  19. Park SY, Lee SJ, Oh TK, Oh JW, Koo BT, Yum DY, et al. 2003. AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria. Microbiology 149: 1541-1550.
    Pubmed CrossRef
  20. Parsek MR, Greenberg EP. 2000. Acyl-homoserine lactone quorum sensing in gram-negative bacteria: a signaling mechanism involved in associations with higher organisms. Proc. Natl. Acad. Sci. USA 97: 8789-8793.
    Pubmed PMC CrossRef
  21. Peleg AY, Seifert H, Paterson DL. 2008. Acinetobacter baumannii:emergence of a successful pathogen. Clin. Microbiol. Rev. 21:538-582.
    Pubmed PMC CrossRef
  22. Rasmussen TB, Givskov M. 2006. Quorum-sensing inhibitors as anti-pathogenic drugs. Int. J. Med. Microbiol. 296: 149-161.
    Pubmed CrossRef
  23. Rutherford ST, Bassler BL. 2012. Bacterial quorum sensing:its role in virulence and possibilities for its control. Cold Spring Harb. Perspect. Med. 2: a012427.
    Pubmed PMC CrossRef
  24. Shrout JD, Chopp DL, Just CL, Hentzer M, Givskov M, Parsek MR. 2006. The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional. Mol. Microbiol. 62: 1264-1277.
    Pubmed CrossRef
  25. Simon R, Priefer U, Pühler A. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Nat. Biotechnol. 1:784-791.
    CrossRef
  26. Stacy DM, Welsh MA, Rather PN, Blackwell HE. 2012. Attenuation of quorum sensing in the pathogen Acinetobacter baumannii using non-native N-acyl homoserine lactones. ACS Chem. Biol. 7: 1719-1728.
    Pubmed PMC CrossRef
  27. Thompson RJ, Bobay BG, Stowe SD, Olson AL, Peng L, Su Z, et al. 2012. Identification of BfmR, a response regulator involved in biofilm development, as a target for a 2-aminoimidazole-based antibiofilm agent. Biochemistry 51:9776-9778.
    Pubmed PMC CrossRef
  28. Tomaras AP, Dorsey CW, Edelmann RE, Actis LA. 2003. Attachment to and biofilm formation on abiotic surfaces by Acinetobacter baumannii: involvement of a novel chaperoneusher pili assembly system. Microbiology 149: 3473-3484.
    Pubmed CrossRef
  29. Tucker AT, Nowicki EM, Boll JM, Knauf GA, Burdis NC, Trent MS, et al. 2014. Defining gene-phenotype relationships in Acinetobacter baumannii through one-step chromosomal gene inactivation. mBio 5: e01313-e01314.
    Pubmed PMC CrossRef
  30. Zhang L, Murphy PJ, Kerr A, Tate ME. 1993. Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones. Nature 362: 446-448.
    Pubmed CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2015; 25(8): 1390-1400

Published online August 28, 2015 https://doi.org/10.4014/jmb.1504.04069

Copyright © The Korean Society for Microbiology and Biotechnology.

Role of LuxIR Homologue AnoIR in Acinetobacter nosocomialis and the Effect of Virstatin on the Expression of anoR Gene

Man Hwan Oh 1 and Chul Hee Choi 2*

1Department of Nanobiomedical Science, Dankook University, Cheonan 330-714, Republic of Korea, 2Department of Microbiology and Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Daejeon 301-747, Republic of Korea

Received: April 27, 2015; Accepted: May 14, 2015

Abstract

Quorum sensing is a process of cell-to-cell communication in which bacteria produce
autoinducers as signaling molecules to sense cell density and coordinate gene expression. In
the present study, a LuxI-type synthase, AnoI, and a LuxR-type regulator, AnoR, were
identified in Acinetobacter nosocomialis, an important nosocomial pathogen, by sequence
analysis of the bacterial genome. We found that N-(3-hydroxy-dodecanoyl)-L-homoserine
lactone (OH-dDHL) is a quorum-sensing signal in A. nosocomialis. The anoI gene deletion was
responsible for the impairment in the production of OH-dDHL. The expression of anoI was
almost abolished in the anoR mutant. These results indicate that AnoI is essential for the
production of OH-dDHL in A. nosocomialis, and its expression is positively regulated by AnoR.
Moreover, the anoR mutant exhibited deficiency in biofilm formation. In particular, motility of
the anoR mutant was consistently and significantly abolished compared with that of the wild
type. The deficiency in the biofilm formation and motility of the anoR mutant was significantly
restored by a functional anoR, indicating that AnoR plays important roles in the biofilm
formation and motility. Furthermore, the present study showed that virstatin exerts its effects
on the reduction of biofilm formation and motility by inhibiting the expression of anoR.
Consequently, the combined results suggest that AnoIR is a quorum-sensing system that plays
important roles in the biofilm formation and motility of A. nosocomialis, and virstatin is an
inhibitor of the expression of anoR.

Keywords: Acinetobacter nosocomialis, quorum sensing, AnoIR, biofilm, motility, virstatin

References

  1. Bhargava N, Sharma P, Capalash N. 2010. Quorum sensing in Acinetobacter: an emerging pathogen. Crit. Rev. Microbiol. 36: 349-360.
    Pubmed CrossRef
  2. Callahan SM, Dunlap PV. 2000. LuxR- and acyl-homoserinelactonecontrolled non-lux genes define a quorum-sensing regulon in Vibrio fischeri. J. Bacteriol. 182: 2811-2822.
    Pubmed KoreaMed CrossRef
  3. Chabane YN, M louka M B, A lexandre S, Nicol M, M arti S, Pestel-Caron M, et al. 2014. Virstatin inhibits biofilm formation and motility of Acinetobacter baumannii. BMC Microbiol. 14: 62.
    Pubmed KoreaMed CrossRef
  4. Chow JY, Yang Y, Tay SB, Chua KL, Yew WS. 2014. Disruption of biofilm formation by the human pathogen Acinetobacter baumannii using engineered quorum-quenching lactonases. Antimicrob. Agents Chemother. 58: 1802-1805.
    Pubmed KoreaMed CrossRef
  5. Clemmer KM, Bonomo RA, Rather PN. 2011. Genetic analysis of surface motility in Acinetobacter baumannii. Microbiology 157: 2534-2544.
    Pubmed KoreaMed CrossRef
  6. Cook DM, Li P-L, Ruchaud F, Padden S, Farrand SK. 1997. Ti plasmid conjugation is independent of vir: reconstitution of the tra functions from pTiC58 as a binary system. J. Bacteriol. 179: 1291-1297.
    Pubmed KoreaMed CrossRef
  7. Dijkshoorn L, Nemec A, Seifert H. 2007. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat. Rev. Microbiol. 5: 939-951.
    Pubmed CrossRef
  8. Gaddy JA, Tomaras AP, Actis LA. 2009. The Acinetobacter baumannii 19606 OmpA protein plays a role in biofilm formation on abiotic surfaces and in the interaction of this pathogen with eukaryotic cells. Infect. Immun. 77: 3150-3160.
    Pubmed KoreaMed CrossRef
  9. Gray KM, Passador L, Iglewski BH, Greenberg EP. 1994. Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa. J. Bacteriol. 176: 3076-3080.
    Pubmed KoreaMed CrossRef
  10. Hall-Stoodley L, Costerton JW, Stoodley P. 2004. Bacterial biofilms: from the natural environment to infectious diseases. Nat. Rev. Microbiol. 2: 95-108.
    Pubmed CrossRef
  11. Harshey RM. 2003. Bacterial motility on a surface: many ways to a common goal. Annu. Rev. Microbiol. 57: 249-273.
    Pubmed CrossRef
  12. Hung DT, Shakhnovich EA, Pierson E, Mekalanos JJ. 2005. Small-molecule inhibitor of Vibrio cholerae virulence and intestinal colonization. Science 310: 670-674.
    Pubmed CrossRef
  13. Hunger M, Schmucker R, Kishan V, Hillen W. 1990. Analysis and nucleotide sequence of an origin of DNA replication in Acinetobacter calcoaceticus and its use for Escherichia coli shuttle plasmids. Gene 87: 45-51.
    CrossRef
  14. Kim J, Park W. 2013. Identification and characterization of genes regulated by AqsR, a LuxR-type regulator in Acinetobacter oleivorans DR1. Appl. Microbiol. Biotechnol. 97: 6967-6978.
    Pubmed CrossRef
  15. Lee YC, Huang YT, Tan CK, Kuo YW, Liao CH, Lee PI, et al. 2011. Acinetobacter baumannii and Acinetobacter genospecies 13TU and 3 bacteraemia: comparison of clinical features, prognostic factors and outcomes. J. Antimicrob. Chemother. 66: 1839-1846.
    Pubmed CrossRef
  16. Nemec A, Krizova L, Maixnerova M, van der Reijden TJ, Deschaght P, Passet V, et al. 2011. Genotypic and phenotypic characterization of the Acinetobacter calcoaceticus–Acinetobacter baumannii complex with the proposal of Acinetobacter pittii sp. nov. (formerly Acinetobacter genomic species 3) and Acinetobacter nosocomialis sp. nov. (formerly Acinetobacter genomic species 13TU). Res. Microbiol. 162: 393-404.
    Pubmed CrossRef
  17. Niu C, Clemmer KM, Bonomo RA, Rather PN. 2008. Isolation and characterization of an autoinducer synthase from Acinetobacter baumannii. J. Bacteriol. 190: 3386-3392.
    Pubmed KoreaMed CrossRef
  18. Oh MH, Lee JC, Kim J, Choi CH, Han K. 2015. Simple method for markerless gene deletion of multidrug-resistant Acinetobacter baumannii. Appl. Environ. Microbiol. 81: 3357-3368.
    Pubmed KoreaMed CrossRef
  19. Park SY, Lee SJ, Oh TK, Oh JW, Koo BT, Yum DY, et al. 2003. AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria. Microbiology 149: 1541-1550.
    Pubmed CrossRef
  20. Parsek MR, Greenberg EP. 2000. Acyl-homoserine lactone quorum sensing in gram-negative bacteria: a signaling mechanism involved in associations with higher organisms. Proc. Natl. Acad. Sci. USA 97: 8789-8793.
    Pubmed KoreaMed CrossRef
  21. Peleg AY, Seifert H, Paterson DL. 2008. Acinetobacter baumannii:emergence of a successful pathogen. Clin. Microbiol. Rev. 21:538-582.
    Pubmed KoreaMed CrossRef
  22. Rasmussen TB, Givskov M. 2006. Quorum-sensing inhibitors as anti-pathogenic drugs. Int. J. Med. Microbiol. 296: 149-161.
    Pubmed CrossRef
  23. Rutherford ST, Bassler BL. 2012. Bacterial quorum sensing:its role in virulence and possibilities for its control. Cold Spring Harb. Perspect. Med. 2: a012427.
    Pubmed KoreaMed CrossRef
  24. Shrout JD, Chopp DL, Just CL, Hentzer M, Givskov M, Parsek MR. 2006. The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional. Mol. Microbiol. 62: 1264-1277.
    Pubmed CrossRef
  25. Simon R, Priefer U, Pühler A. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Nat. Biotechnol. 1:784-791.
    CrossRef
  26. Stacy DM, Welsh MA, Rather PN, Blackwell HE. 2012. Attenuation of quorum sensing in the pathogen Acinetobacter baumannii using non-native N-acyl homoserine lactones. ACS Chem. Biol. 7: 1719-1728.
    Pubmed KoreaMed CrossRef
  27. Thompson RJ, Bobay BG, Stowe SD, Olson AL, Peng L, Su Z, et al. 2012. Identification of BfmR, a response regulator involved in biofilm development, as a target for a 2-aminoimidazole-based antibiofilm agent. Biochemistry 51:9776-9778.
    Pubmed KoreaMed CrossRef
  28. Tomaras AP, Dorsey CW, Edelmann RE, Actis LA. 2003. Attachment to and biofilm formation on abiotic surfaces by Acinetobacter baumannii: involvement of a novel chaperoneusher pili assembly system. Microbiology 149: 3473-3484.
    Pubmed CrossRef
  29. Tucker AT, Nowicki EM, Boll JM, Knauf GA, Burdis NC, Trent MS, et al. 2014. Defining gene-phenotype relationships in Acinetobacter baumannii through one-step chromosomal gene inactivation. mBio 5: e01313-e01314.
    Pubmed KoreaMed CrossRef
  30. Zhang L, Murphy PJ, Kerr A, Tate ME. 1993. Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones. Nature 362: 446-448.
    Pubmed CrossRef