Advertisement

Upregulation of the clpB gene in response to heat shock and beta-lactam antibiotics in Acinetobacter baumannii

  • Waleska Yana Lazaretti
  • Elaine Luzia dos Santos
  • José Luis da-Conceição Silva
  • Marina Kimiko Kadowaki
  • Rinaldo Ferreira Gandra
  • Alexandre Maller
  • Rita de Cássia Garcia SimãoEmail author
Short Communication

Abstract

The role of the clpB gene encoding HSP/chaperone ClpB was evaluated in the multiresistant antibiotic cells of Acinetobacter baumannii (RS4 strain) under stress-induced heat shock and different beta-lactams. The expression of the clpB gene was assessed by qPCR during heat shock at 45 °C and subinhibitory concentrations of ampicillin (30 μg mL−1), amoxicillin + sulbactam (8/12 μg mL−1), cefepime (30 μg mL−1), sulfamethoxazole + trimethoprim (120/8 μg mL−1) and meropenem (18 μg mL−1). The results indicated a transient increase in clpB transcription in all treatments except cefepime. Both in the presence of ampicillin and amoxicillin/sulbactam for 20 min, the mRNA-clpB synthesis was 1.4 times higher than that of the control at time zero. Surprisingly, the mRNA-clpB levels were more than 30-fold higher after 10 min of incubation with meropenem and more than eightfold higher in the presence of trimethoprim/sulfamethoxazole. In addition, western blot assays showed that the RS4 strain treated with meropenem showed a marked increase in ClpB protein expression. Our data indicate that during exposure to beta-lactams, A. baumannii adjusts the transcription levels of the clpB mRNA and protein to respond to stress, suggesting that the chaperone may act as a key cellular component in the presence of antibiotics in this bacterium.

Keywords

ClpB Stress response Antibiotics Heat shock proteins Multidrug resistance 

Notes

Funding

W. Y. Lazaretti is a professional funded by the Brazilian National Health Surveillance Agency (ANVISA). E. L. Santos was a fellow of the Brazilian National Council for Scientific and Technological Development (CNPq).

Compliance with ethical standards

Conflict of interest

All the authors declared that they have no conflict of interest.

Ethical approval

The authors declare that the research was performed in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary material

11033_2019_5209_MOESM1_ESM.pptx (2.9 mb)
Supplementary material 1 (PPTX 3013 kb). Fig. S1. Agarose gel electrophoresis showing the products of RNA extraction obtained from the heat shock treatments and exposure to antibiotics of the multidrug resistant A. baumannii RS4 strain. (A) Heat shock at 45 °C, (B) meropenem, (C) cefepime, (D) sulfamethoxazole + trimethoprim, (E) ampicillin, (F) amoxicillin + sulbactam. (T0) time zero, (T10) 10 min, (T20) 20 min and (T30) 30 min

References

  1. 1.
    Maragakis LL, PerL TM (2008) Acinetobacter baumannii: epidemiology, antimicrobial resistance and treatment options. Clin Infect Dis 46(8):1254–1263.  https://doi.org/10.1086/529.198 CrossRefPubMedGoogle Scholar
  2. 2.
    Gebhardt MJ, Gallagher LA, Jacobson RK, Usacheva EA, Peterson LR, Zurawski DV et al (2015) Joint transcriptional control of virulence and resistance to antibiotic and environmental stress in Acinetobacter baumannii. MBio 6:e01660.  https://doi.org/10.1128/mBio.01660-15 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Rossi I, Royer S, Ferreira ML, Campos PA, Fuga B, Melo GN, Machado LG, Resende DS, Batistão D, Urzedo JE, Gontijo-Filho PP, Ribas RM (2019) Incidence of infections caused by carbapenem-resistant Acinetobacter baumannii. Am J Infect Control.  https://doi.org/10.1016/j.ajic.2019.07.009 CrossRefPubMedGoogle Scholar
  4. 4.
    Falagas ME, Bliziotis IA, Siempos II (2006) Attributable mortality of Acinetobacter baumannii infections in critically ill patients: a systematic review of matched cohort and case-control studies. Crit Care 10:48.  https://doi.org/10.1186/cc4869 CrossRefGoogle Scholar
  5. 5.
    Kempf M, Rolain JM (2012) Emergence of resistance to carbapenems in Acinetobacter baumannii in Europe: clinical impact and therapeutic options. Int J Antimicrob Agents 39:105–114.  https://doi.org/10.1016/j.ijantimicag.2011.10.004 CrossRefPubMedGoogle Scholar
  6. 6.
    Qureshi ZA, Hittle LE, Ohara JA, Rivera JI, Syed A, Shields RK et al (2015) Colistin-resistant Acinetobacter baumannii: beyond carbapenem resistance. Clin Infect Dis 60:1295–1303.  https://doi.org/10.1093/cid/civ048 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Gomes SL, Simao RCG (2014) Heat: stresses responses. In: Michael Caplan (Ed-in chief)/Ralph Bradshaw (subject Ed.) (Or.) In Reference Module in Biomedical Sciences - Microbial Physiology and Metabolism. Elsevier, vol 1, pp 1–16Google Scholar
  8. 8.
    Yamaguchi Y, Tomoyasu T, Takaya A, Morioka M, Yamamoto T (2003) Effects of disruption of heat shock genes on susceptibility of Escherichia coli to fluoroquinolones. BMC Microbiol 3:16.  https://doi.org/10.1186/1471-2180-3-16 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Cardoso K, Gandra RF, Wisniewski ES, Osaku CA, Kadowaki MK, Felipach-Neto V et al (2010) DnaK and GroEL are induced in response to antibiotic and heat shock in Acinetobacter baumannii. J Med Microbiol 59:1061–1068.  https://doi.org/10.1099/jmm.0.020339-0 CrossRefPubMedGoogle Scholar
  10. 10.
    Wong P, Houry WA (2004) Chaperone networks in bacteria: analysis of protein homeostasis in minimal cells. J Struct Biol 146:79–89.  https://doi.org/10.1016/j.jsb.2003.11.006 CrossRefPubMedGoogle Scholar
  11. 11.
    Clauss-Lendzian E, Vaishampayan A, De Jong A, Landau U, Meyer C, Kok J et al (2018) Stress response of a clinical Enterococcus faecalis isolate subjected to a novel antimicrobial surface coating. Microbiol Res 207:53–64.  https://doi.org/10.1016/j.micres.2017.11.006 CrossRefPubMedGoogle Scholar
  12. 12.
    Gophna U, Ron EZ (2003) Virulence and the heat shock response. Int J Med Microbiol 292:453–461.  https://doi.org/10.1078/1438-4221-00230 CrossRefPubMedGoogle Scholar
  13. 13.
    Frees D, Gerth U, Ingmer H (2014) Clp chaperones and proteases are central in stress survival, virulence and antibiotic resistance of Staphylococcus aureus. Int J Med Microbiol 304:142–149.  https://doi.org/10.1016/j.ijmm.2013.11.009 CrossRefPubMedGoogle Scholar
  14. 14.
    Scribano D, Marzano V, Mortera SL, Sarshar M, Vernocchi P, Zagaglia C, Putignani L, Palamara AT, Ambrosi C (2019) Insights into the periplasmic proteins of Acinetobacter baumannii AB5075 and the impact of imipenem exposure: a proteomic approach. Int J Mol Sci 20:3451.  https://doi.org/10.3390/ijms20143451 CrossRefPubMedCentralGoogle Scholar
  15. 15.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  16. 16.
    Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622.  https://doi.org/10.1373/clinchem.2008.112797 CrossRefPubMedGoogle Scholar
  17. 17.
    Sharma R, Arya S, Patil SD, Sharma A, Jain PK, Navani NK et al (2014) Identification of novel regulatory small RNAs in Acinetobacter baumannii. PLoS ONE 9:e93833.  https://doi.org/10.1371/journal.pone.0093833 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  19. 19.
    Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 76:4350–4354CrossRefGoogle Scholar
  20. 20.
    Simão RC, Susin MF, Alvarez-Martinez CE, Gomes SL (2005) Cells lacking ClpB display a prolonges shutoff phase of the heat shock response in Caulobacter crescentus. Mol Microbiol 57:592–603.  https://doi.org/10.1111/j.1365-2958.2005.04713.x CrossRefPubMedGoogle Scholar
  21. 21.
    De Oliveira NEM, Abranches J, Gaca AO, Laport MS, Damaso CR, De Freire Bastos MDC et al (2011) ClpB, a class III heat-shock gene regulated by CtsR, is involved in thermotolerance and virulence of Enterococcus faecalis. Microbiology 157:656–665.  https://doi.org/10.1099/mic.0.041897-0 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Chastanet A, Derre I, Nair S, Msadek T (2004) ClpB, a novel member of the Listeria monocytogenes CtsR regulon, is involved in virulence but not in general stress tolerance. J Bacteriol 186:1165–1174.  https://doi.org/10.1128/JB.186.4.1165-1174.2004 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Montagu A, Joly-Guillou ML, Rossines E, Cayon J, Kempf M, Saulnier P (2016) Stress conditions induced by carvacrol and cinnamaldehyde on Acinetobacter baumannii. Front Microbiol 7:1133.  https://doi.org/10.3389/fmicb.2016.01133 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Waleska Yana Lazaretti
    • 1
  • Elaine Luzia dos Santos
    • 1
  • José Luis da-Conceição Silva
    • 1
  • Marina Kimiko Kadowaki
    • 1
  • Rinaldo Ferreira Gandra
    • 2
  • Alexandre Maller
    • 1
  • Rita de Cássia Garcia Simão
    • 1
    Email author
  1. 1.Laboratório de Bioquímica Molecular, Centro de Ciências Médicas e FarmacêuticasUniversidade Estadual do Oeste do Paraná, UNIOESTECascavelBrazil
  2. 2.Laboratório de Micologia Clínica, Hospital Universitário do Oeste do ParanáUniversidade Estadual do Oeste do Paraná, UNIOESTECascavelBrazil

Personalised recommendations