J Bacteriol Virol.  2020 Jun;50(2):97-106. 10.4167/jbv.2020.50.2.097.

Effects of Exogenous N-Acyl-Homoserine Lactones on Biofilm Formation and Motility in Acinetobacter nosocomialis

• Surendran S
• Subhadra B
• Woo K
• Park HS
• Kim DH
• Oh MH
• Choi CH

• KMID: 2504385
• DOI: http://doi.org/10.4167/jbv.2020.50.2.097

Abstract

One of the major factors contributing to drug resistance in Acinetobacter nosocomialis infections is biofilm development, which is facilitate by quorum-sensing (QS) systems. Quorum sensing by the LuxI and LuxR homologues, AnoI and AnoR, in A. nosocomialis plays a role in biofilm formation and motility of this pathogenic bacterium. The aim of this study was to evaluate the effects of exogenous N-acyl-homoserine lactones (AHLs) on the regulation of biofilm and motility of A. nosocomialis and anoI-deletion mutant. We found that anoR mRNA expression levels in the anoI-deletion mutant were increased in the presence of different types of AHLs compared with that in the absence of exogenous AHL. Among AHLs, C12-HSL appeared to exert the greatest stimulatory effect on biofilm formation and motility. Notably, the anoI-deletion mutant also exhibited significant decreases in expression of the biofilm- and motility-related genes, csuC, csuD and pilT, decreases that were attenuated by addition of exogenous AHLs. Combining the AHL C12-HSL with C6-HSL or C10-HSL exerted synergistic effects that restored the motility phenotype in the anoI-deletion mutant. Taken together, our data demonstrate that C12-HSL may act as an important signaling molecule in A. nosocomialis through regulation of biofilm formation and cell motility, potentially providing a new target for the control of A. nosocomialis infections.

Keyword

Quorum sensing; Exogenous AHLs; Biofilm formation; Motility; A. nosocomialis

Figure

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  Fig. 1 Confirmation of the anoI-deletion mutant. (A) Gel image showing PCR analysis of the WT and anoI-deletion mutant (∆anoI), confirming deletion of the target gene. (B) Representative growth curves for the WT and anoI-deletion mutant. (C) anoI mRNA expression was nearly absent in the anoI-deletion mutant. ***P < 0.001 versus WT.

• 

  Fig. 2 Relative mRNA expression of anoR in the absence and presence of the indicated concentration (40 and 80 mM) and type of AHL. (A–D) anoR mRNA expression in the WT and anoI-deletion mutant following exposure to different concentrations of C6-HSL (A), C10-HSL (B) or C12-HSL (C). The differences among different sample groups were evaluated using a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test using Graph-Pad Prism Software. ***P < 0.001 versus the anoI-deletion mutant.

• 

  Fig. 3 Biofilm formation and motility. (A) Crystal violet staining of biofilm formed by the WT or anoI-deletion mutant in the presence (40 μM) and absence of exogenous AHLs. (B) Graphical representation of biofilm formation, quantified as absorbance at OD590 nm. The differences among different sample groups were evaluated using a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test using Graph-Pad Prism Software. (C) Motility assay of the WT in the absence of exogenous AHLs and of the anoI-deletion mutant in both the absence and presence of AHLs. **P < 0.01 and ***P < 0.001 versus the anoI-deletion mutant.

• 

  Fig. 4 Relative mRNA expression of various genes related to biofilm formation and motility. Expression of csuC, csuD and pilT genes in the WT and anoI-deletion mutant strain in the presence (40 μM) and absence of exogenous AHLs, measured by qRT-PCR. The differences among different sample groups were evaluated using a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test using Graph-Pad Prism Software. *P < 0.05, **P < 0.01 and ***P < 0.001 versus the anoI-deletion mutant.

Reference

1. Qi C, Malczynski M, Parker M, Scheetz MH. Characterization of genetic diversity of carbapenem-resistant Acinetobacter baumannii clinical strains collected from 2004 to 2007. J Clin Microbiol 2008;46:1106-9.DOI: 10.1128/JCM.01877-07. PMID: 18216212. PMCID: PMC2268351.

2. Wang X, Chen T, Yu R, Lü X, Zong Z. Acinetobacter pittii and Acinetobacter nosocomialis among clinical isolates of the Acinetobacter calcoaceticus-baumannii complex in Sichuan, China. Diagn Microbiol Infect Dis 2013;76:392-5.DOI: 10.1016/j.diagmicrobio.2013.03.020. PMID: 23639796.

3. Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev 2008;21:538-82.DOI: 10.1128/CMR.00058-07. PMID: 18625687. PMCID: PMC2493088.

4. Withers H, Swift S, Williams P. Quorum sensing as an integral component of gene regulatory networks in Gram-negative bacteria. Curr Opin Microbiol 2001;4:186-93.DOI: 10.1016/S1369-5274(00)00187-9.

5. Bassler BL, Wright M, Showalter RE, Silverman MR. Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Mol Microbiol 1993;9:773-86.DOI: 10.1111/j.1365-2958.1993.tb01737.x. PMID: 8231809.

6. Miller MB, Bassler BL. Quorum sensing in bacteria. Annu Rev Microbiol 2001;55:165-99.DOI: 10.1146/annurev.micro.55.1.165. PMID: 11544353.

7. Oh MH, Choi CH. Role of LuxIR Homologue AnoIR in Acinetobacter nosocomialis and the Effect of Virstatin on the Expression of anoR Gene. J Microbiol Biotechnol 2015;25:1390-400.DOI: 10.4014/jmb.1504.04069. PMID: 25975610.

8. Zhu YL, Hou HM, Zhang GL, Wang YF, Hao HS. AHLs Regulate Biofilm Formation and Swimming Motility of Hafnia alvei H4. Front Microbiol 2019;10:1330.DOI: 10.3389/fmicb.2019.01330. PMID: 31275267. PMCID: PMC6593095.

9. Lee HW, Koh YM, Kim J, Lee JC, Lee YC, Seol SY, et al. Capacity of multidrug-resistant clinical isolates of Acinetobacter baumannii to form biofilm and adhere to epithelial cell surfaces. Clin Microbiol Infect 2008;14:49-54.DOI: 10.1111/j.1469-0691.2007.01842.x. PMID: 18005176.

10. Armbruster CR, Parsek MR. New insight into the early stages of biofilm formation. Proc Natl Acad Sci USA 2018;115:4317-9.DOI: 10.1073/pnas.1804084115. PMID: 29632199. PMCID: PMC5924939.

11. Tolker-Nielsen T. Biofilm Development. Microbiol Spectr 2015;3:MB-0001-2014.DOI: 10.1128/microbiolspec.MB-0001-2014. PMID: 26104692.

12. Dunne WM Jr. Bacterial adhesion: seen any good biofilms lately? Clin Microbiol Rev 2002;15:155-66.DOI: 10.1128/CMR.15.2.155-166.2002. PMID: 11932228. PMCID: PMC118072.

13. Fux CA, Costerton JW, Stewart PS, Stoodley P. Survival strategies of infectious biofilms. Trends Microbiol 2005;13:34-40.DOI: 10.1016/j.tim.2004.11.010. PMID: 15639630.

14. Nemec A, Krizova L, Maixnerova M, van der Reijden TJ, Deschaght P, Passet V, et al. 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 2011;162:393-404.DOI: 10.1016/j.resmic.2011.02.006. PMID: 21320596.

15. Oh MH, Lee JC, Kim J, Choi CH, Han K. Simple Method for Markerless Gene Deletion in Multidrug-Resistant Acinetobacter baumannii. Appl Environ Microbiol 2015;81:3357-68.DOI: 10.1128/AEM.03975-14. PMID: 25746991. PMCID: PMC4407223.

16. Eberl L. N-acyl homoserinelactone-mediated gene regulation in gram-negative bacteria. Syst Appl Microbiol 1999;22:493-506.DOI: 10.1016/S0723-2020(99)80001-0.

17. Saipriya K, Swathi CH, Ratnakar KS, Sritharan V. Quorum-sensing system in Acinetobacter baumannii: a potential target for new drug development. J Appl Microbiol 2020;128:15-27.DOI: 10.1111/jam.14330. PMID: 31102552.

18. Rasmussen TB, Givskov M. Quorum sensing inhibitors: a bargain of effects. Microbiology 2006;152:895-904.DOI: 10.1099/mic.0.28601-0. PMID: 16549654.

19. Li Z, Nair SK. Quorum sensing: how bacteria can coordinate activity and synchronize their response to external signals? Protein Sci 2012;21:1403-17.DOI: 10.1002/pro.2132. PMID: 22825856. PMCID: PMC3526984.

20. Rémy B, Mion S, Plener L, Elias M, Chabrière E, Daudé D. Interference in Bacterial Quorum Sensing: A Biopharmaceutical Perspective. Front Pharmacol 2018;9:203.DOI: 10.3389/fphar.2018.00203. PMID: 29563876. PMCID: PMC5845960.

21. Gurich N, González JE. Role of quorum sensing in Sinorhizobium meliloti-Alfalfa symbiosis. J Bacteriol 2009;191:4372-82.DOI: 10.1128/JB.00376-09. PMID: 19395488. PMCID: PMC2698488.

22. Andersson RA, Eriksson AR, Heikinheimo R, Mäe A, Pirhonen M, Kõiv V, et al. Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora: the role of expR (Ecc). Mol Plant Microbe Interact 2000;13:384-93.DOI: 10.1094/MPMI.2000.13.4.384. PMID: 10755301.

23. Nasser W, Bouillant ML, Salmond G, Reverchon S. Characterization of the Erwinia chrysanthemi expI-expR locus directing the synthesis of two N-acyl-homoserine lactone signal molecules. Mol Microbiol 1998;29:1391-405.DOI: 10.1046/j.1365-2958.1998.01022.x. PMID: 9781877.