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The character of PA3235 virulence factors of Pseudomonas aeruginosa PAO1 – a preliminary study

  • Mariana Wahjudi ,
  • Samuel Stefanus Widodo ,
  • Ida Bagus Made Artadana ,
  • Yulanda Antonius ,


Abstract

Introduction: Many virulence factors of Pseudomonas aeruginosa PAO1 are regulated by temperature and host conditions upon infection. Based on microarray data, the PA3235 gene is one of the upregulated genes during cell growth at 37 °C. Until now, no information about its role in PAO1 pathogenicity.

Methods: The PAO1∆PA3235 strain was constructed by overlapping polymerase chain reaction (PCR) method and through biparental mating. The deletion was confirmed by PCR and restriction analyses. The virulence factors of both P. aeruginosa PAO1 wild type and ∆PA3235 mutant strains were examined, which consisted of the amount of pyocyanin and pyoverdine, swarming, swimming, and twitching motility, biofilm formation, 3-oxo-dodecanoyl-homoserine lactone concentration, and growth curve profile. Data were analyzed using Student’s t-tests to determine differences between treatments. P-value < 0.05 were considered significant.

Results: The ∆PA3235 strain was successfully constructed. At 37 ºC, the mutant produced less pyocyanin (p-value 0.0004), pyoverdine (p-value 0.0009), and swarm area than the wild-type. The dendrites pattern of both strains was similar. The mutant and parental strains showed no differences in swimming and twitching motility when incubated at 22 ºC and 37 ºC. The mutant produced more biofilm compared to the wild-type strain (p-value 0.0013). The AHL was higher in the mutant than in wild type strain (p-value 0.0095) after 24 h incubation. Both the wild type and mutant strains exhibited similar growth patterns in LB broth. The mutant colonies also showed the same morphology as the wild type on the LB plate (not shown here).

Conclusion: The deletion of the PA3235 gene from the Pseudomonas aeruginosa genome caused some changes in virulence factors production, as the bacterium grew at body temperature 37°C. We predicted that the PA3235 gene might function to transport molecules involved in the early infection of this bacterium to humans.

Keywords: pathogenicity pyocyanin pyoverdine swarming swimming twitching

References

  1. Paulsson M, Su YC, Ringwood T, Uddén F, Riesbeck K. Pseudomonas aeruginosa uses multiple receptors for adherence to laminin during infection of the respiratory tract and skin wounds. Sci Rep. 2019;9(1):18168. Published 2019 Dec 3. doi:10.1038/s41598-019-54622-z.
  2. Chahtane H, Nogueira Füller T, Allard PM, et al. The plant pathogen Pseudomonas aeruginosa triggers a DELLA-dependent seed germination arrest in Arabidopsis. Elife. 2018;7:e37082. Published 2018 Aug 28. doi:10.7554/eLife.37082.
  3. Bachta KER, Allen JP, Cheung BH, Chiu CH & Hauser AR. Systemic infection facilitates transmission of Pseudomonas aeruginosa in mice. Nat Commun. 2020;11(543). doi: 10.1038/s41467-020-14363-4.
  4. Stover CK, Pham XQ, Erwin AL, et al. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature. 2000;406(6799):959-964. doi:10.1038/35023079.
  5. Klockgether J, Cramer N, Wiehlmann L, Davenport CF, Tümmler B. Pseudomonas aeruginosa Genomic Structure and Diversity. Front Microbiol. 2011;2:150. Published 2011 Jul 13. doi:10.3389/fmicb.2011.00150.
  6. Wurtzel O, Yoder-Himes DR, Han K, et al. The single-nucleotide resolution transcriptome of Pseudomonas aeruginosa grown in body temperature. PLoS Pathog. 2012;8(9):e1002945. doi:10.1371/journal.ppat.1002945.
  7. Kruczek C, Kottapalli KR, Dissanaike S, et al. Major Transcriptome Changes Accompany the Growth of Pseudomonas aeruginosa in Blood from Patients with Severe Thermal Injuries. PLoS One. 2016;11(3):e0149229. Published 2016 Mar 2. doi:10.1371/journal.pone.0149229.
  8. Beasley KL, Cristy SA, Elmassry MM, Dzvova N, Colmer-Hamood JA, Hamood AN. During bacteremia, Pseudomonas aeruginosa PAO1 adapts by altering the expression of numerous virulence genes including those involved in quorum sensing. PLoS One. 2020;15(10):e0240351. Published 2020 Oct 15. doi:10.1371/journal.pone.0240351.
  9. Moradali MF, Ghods S, Rehm BH. Pseudomonas aeruginosa Lifestyle: A Paradigm for Adaptation, Survival, and Persistence. Front Cell Infect Microbiol. 2017;7:39. Published 2017 Feb 15. doi:10.3389/fcimb.2017.00039.
  10. Barbier M, Damron FH, Bielecki P, et al. From the environment to the host: re-wiring of the transcriptome of Pseudomonas aeruginosa from 22°C to 37°C. PLoS One. 2014;9(2):e89941. Published 2014 Feb 24. doi:10.1371/journal.pone.0089941.
  11. Bielecki P, Komor U, Bielecka A, et al. Ex vivo transcriptional profiling reveals a common set of genes important for the adaptation of Pseudomonas aeruginosa to chronically infected host sites. Environ Microbiol. 2013;15(2):570-587. doi:10.1111/1462-2920.12024.
  12. Lewenza S, Gardy JL, Brinkman FS, Hancock RE. Genome-wide identification of Pseudomonas aeruginosa exported proteins using a consensus computational strategy combined with a laboratory-based PhoA fusion screen. Genome Res. 2005;15(2):321-329. doi:10.1101/gr.3257305.
  13. Marchler-Bauer A, Bo Y, Han L, et al. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 2017;45(D1):D200-D203. doi:10.1093/nar/gkw1129.
  14. Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP. A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene. 1998;212(1):77-86. doi:10.1016/s0378-1119(98)00130-9.
  15. Simon R, Quandt J, Klipp W. New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria. Gene. 1989;80(1):161-169. doi:10.1016/0378-1119(89)90262-x.
  16. Essar DW, Eberly L, Hadero A, Crawford IP. Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications. J Bacteriol. 1990;172(2):884-900. doi:10.1128/jb.172.2.884-900.1990.
  17. Diggle SP, Winzer K, Lazdunski A, Williams P, Cámara M. Advancing the quorum in Pseudomonas aeruginosa: MvaT and the regulation of N-acylhomoserine lactone production and virulence gene expression. J Bacteriol. 2002;184(10):2576-2586. doi:10.1128/JB.184.10.2576-2586.2002.
  18. Atkinson S, Chang CY, Sockett RE, Cámara M, Williams P. Quorum sensing in Yersinia enterocolitica controls swimming and swarming motility. J Bacteriol. 2006;188(4):1451-1461. doi:10.1128/JB.188.4.1451-1461.2006.
  19. Merritt JH, Kadouri DE, O'Toole GA. Growing and analyzing static biofilms. Curr Protoc Microbiol. 2005;Chapter 1:Unit-1B.1. doi:10.1002/9780471729259.mc01b01s00.
  20. Winson MK, Swift S, Fish L, et al. Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acyl homoserine lactone-mediated quorum sensing. FEMS Microbiol Lett. 1998;163(2):185-192. doi:10.1111/j.1574-6968.1998.tb13044.x.
  21. Wahjudi M, Papaioannou E, Hendrawati O, et al. PA0305 of Pseudomonas aeruginosa is a quorum quenching acylhomoserine lactone acylase belonging to the Ntn hydrolase superfamily. Microbiology (Reading). 2011;157(7):2042-2055. doi:10.1099/mic.0.043935-0.
  22. Girard G, Bloemberg GV. Central role of quorum sensing in regulating the production of pathogenicity factors in Pseudomonas aeruginosa. Future Microbiol. 2008;3(1):97-106. doi:10.2217/17460913.3.1.97.
  23. Veetilvalappil VV, Manuel A, Aranjani JM, Tawale R, Koteshwara A. Pathogenic arsenal of Pseudomonas aeruginosa: an update on virulence factors. Future Microbiol. 2022;17:465-481. doi:10.2217/fmb-2021-0158.
  24. Wu D. Genomic analysis and temperature-dependent transcriptome profiles of the rhizosphere originating strain Pseudomonas aeruginosa M18. BMC Genomics. 2011;12:1–17.
  25. Wagner VE, Bushnell D, Passador L, Brooks AI, Iglewski BH. Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment. J Bacteriol. 2003;185(7):2080-2095. doi:10.1128/JB.185.7.2080-2095.2003.
  26. Lequette Y, Lee JH, Ledgham F, Lazdunski A, Greenberg EP. A distinct QscR regulon in the Pseudomonas aeruginosa quorum-sensing circuit. J Bacteriol. 2006;188(9):3365-3370. doi:10.1128/JB.188.9.3365-3370.2006.
  27. Mentel M, Ahuja EG, Mavrodi DV, Breinbauer R, Thomashow LS, Blankenfeldt W. Of two make one: the biosynthesis of phenazines. Chembiochem. 2009;10(14):2295-2304. doi:10.1002/cbic.200900323.
  28. Huang J, Xu Y, Zhang H, et al. Temperature-dependent expression of phzM and its regulatory genes lasI and ptsP in rhizosphere isolate Pseudomonas sp. strain M18. Appl Environ Microbiol. 2009;75(20):6568-6580. doi:10.1128/AEM.01148-09.
  29. Jimenez PN, Koch G, Papaioannou E, et al. Role of PvdQ in Pseudomonas aeruginosa virulence under iron-limiting conditions. Microbiology (Reading). 2010;156(1):49-59. doi:10.1099/mic.0.030973-0.
  30. Burrows LL. Pseudomonas aeruginosa twitching motility: type IV pili in action. Annu Rev Microbiol. 2012;66:493-520. doi:10.1146/annurev-micro-092611-150055.
  31. Köhler T, Curty LK, Barja F, van Delden C, Pechère JC. Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. J Bacteriol. 2000;182(21):5990-5996. doi:10.1128/JB.182.21.5990-5996.2000.
  32. Overhage J, Bains M, Brazas MD, Hancock RE. Swarming of Pseudomonas aeruginosa is a complex adaptation leading to increased production of virulence factors and antibiotic resistance. J Bacteriol. 2008;190(8):2671-2679. doi:10.1128/JB.01659-07.
  33. Tremblay J, Déziel E. Gene expression in Pseudomonas aeruginosa swarming motility. BMC Genomics. 2010;11:587. Published 2010 Oct 20. doi:10.1186/1471-2164-11-587.
  34. Cai Y. Differential impact on motility and biofilm dispersal of closely related phosphodiesterases in Pseudomonas aeruginosa. Sci. Rep. 2020;10:6232.
  35. Anderson GG, Moreau-Marquis S, Stanton BA, O'Toole GA. In vitro analysis of tobramycin-treated Pseudomonas aeruginosa biofilms on cystic fibrosis-derived airway epithelial cells. Infect Immun. 2008;76(4):1423-1433. doi:10.1128/IAI.01373-07.
  36. Thi MTT, Wibowo D, Rehm BHA. Pseudomonas aeruginosa Biofilms. Int J Mol Sci. 2020;21(22):8671. Published 2020 Nov 17. doi:10.3390/ijms21228671.
  37. Chellappa ST, Maredia R, Phipps K, Haskins WE, Weitao T. Motility of Pseudomonas aeruginosa contributes to SOS-inducible biofilm formation. Res Microbiol. 2013;164(10):1019-1027. doi:10.1016/j.resmic.2013.10.001.
  38. Zaitseva J, Granik V, Belik A, Koksharova O, Khmel I. Effect of nitrofurans and NO generators on biofilm formation by Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia 370. Res Microbiol. 2009;160(5):353-357. doi:10.1016/j.resmic.2009.04.007.
  39. Banin E, Vasil ML, Greenberg EP. Iron and Pseudomonas aeruginosa biofilm formation. Proc Natl Acad Sci U S A. 2005;102(31):11076-11081. doi:10.1073/pnas.0504266102.
  40. Caiazza NC, Merritt JH, Brothers KM, O'Toole GA. Inverse regulation of biofilm formation and swarming motility by Pseudomonas aeruginosa PA14. J Bacteriol. 2007;189(9):3603-3612. doi:10.1128/JB.01685-06.
  41. Shrout JD, Chopp DL, Just CL, Hentzer M, Givskov M, Parsek MR. The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional. Mol Microbiol. 2006;62(5):1264-1277. doi:10.1111/j.1365-2958.2006.05421.x.
  42. Krogh A, Larsson B, von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001;305(3):567-580. doi:10.1006/jmbi.2000.4315.
  43. Schuster M, Lostroh CP, Ogi T, Greenberg EP. Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J Bacteriol. 2003;185(7):2066-2079. doi:10.1128/JB.185.7.2066-2079.2003.
  44. Winsor GL, Griffiths EJ, Lo R, Dhillon BK, Shay JA, Brinkman FS. Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database. Nucleic Acids Res. 2016;44(D1):D646-D653. doi:10.1093/nar/gkv1227.
  45. Zheng P, Sun J, Geffers R, Zeng AP. Functional characterization of the gene PA2384 in large-scale gene regulation in response to iron starvation in Pseudomonas aeruginosa. J Biotechnol. 2007;132(4):342-352. doi:10.1016/j.jbiotec.2007.08.013.
  46. van Delden C, Page MG, Köhler T. Involvement of Fe uptake systems and AmpC β-lactamase in susceptibility to the siderophore monosulfactam BAL30072 in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2013;57(5):2095-2102. doi:10.1128/AAC.02474-12.
  47. Kang D, Kirienko NV. Interdependence between iron acquisition and biofilm formation in Pseudomonas aeruginosa. J Microbiol. 2018;56(7):449-457. doi:10.1007/s12275-018-8114-3.
  48. Musk DJ, Banko DA, Hergenrother PJ. Iron salts perturb biofilm formation and disrupt existing biofilms of Pseudomonas aeruginosa. Chem Biol. 2005;12(7):789-796. doi:10.1016/j.chembiol.2005.05.007.
  49. Schmidberger A, Henkel M, Hausmann R, Schwartz T. Influence of ferric iron on gene expression and rhamnolipid synthesis during batch cultivation of Pseudomonas aeruginosa PAO1. Appl Microbiol Biotechnol. 2014;98(15):6725-6737. doi:10.1007/s00253-014-5747-y.
  50. Glick R, Gilmour C, Tremblay J, et al. Increase in rhamnolipid synthesis under iron-limiting conditions influences surface motility and biofilm formation in Pseudomonas aeruginosa. J Bacteriol. 2010;192(12):2973-2980. doi:10.1128/JB.01601-09.
  51. Chugani S, Greenberg EP. The influence of human respiratory epithelia on Pseudomonas aeruginosa gene expression. Microb Pathog. 2007;42(1):29-35. doi:10.1016/j.micpath.2006.10.004.
  52. Cornforth DM, Dees JL, Ibberson CB, et al. Pseudomonas aeruginosa transcriptome during human infection. Proc Natl Acad Sci U S A. 2018;115(22):E5125-E5134. doi:10.1073/pnas.1717525115.
  53. Brindhadevi K. Biofilm and Quorum sensing mediated pathogenicity in Pseudomonas aeruginosa. Process Biochem. 2020; 96:49–57.
  54. Dewi Yana, Widodo, A. D. W., & Pepy Dwi Endraswari. (2023). Comparison between the time to detection of pathogenic and nonpathogenic microorganisms from blood culture specimens using the BACTECTM FX machine to determine the optimal incubation time. Bali Medical Journal, 12(1), 909–915. https://doi.org/10.15562/bmj.v12i1.4199.
  55. Syaiful, I., Widodo, A. D. W., Endraswari, P. D., Alimsardjono, L., Utomo, B., & Arfijanto, M. V. (2023). The association between biofilm formation ability and antibiotic resistance phenotype in clinical isolates of gram-negative bacteria: a cross-sectional study. Bali Medical Journal, 12(1), 1014–1020. https://doi.org/10.15562/bmj.v12i1.4101.
  56. Bramardipa, A. A. B., Sukrama, I. D. M., & Budayanti, N. N. S. (2019). Bacterial pattern and its susceptibility toward antibiotic on burn infection in Burn Unit Sanglah General Hospital. Bali Medical Journal, 8(1), 328–333. https://doi.org/10.15562/bmj.v8i1.1456

How to Cite

Wahjudi, M., Widodo, S. S. ., Artadana, I. B. M. ., & Antonius, Y. . (2023). The character of PA3235 virulence factors of Pseudomonas aeruginosa PAO1 – a preliminary study. Bali Medical Journal, 12(2), 1368–1376. https://doi.org/10.15562/bmj.v12i2.4364
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Mariana Wahjudi
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Samuel Stefanus Widodo
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Ida Bagus Made Artadana
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Yulanda Antonius
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