1887

Abstract

a Gram-negative coccobacillus, is notorious for its involvement in opportunistic infections around the world. Its resistance to antibiotics makes treatment of infections challenging. In this study, we describe a novel response regulator protein, AvnR (A1S_2006) that regulates virulence-related traits in ATCC17978. Sequence analysis suggests that AvnR is a CheY-like response regulator and contains the RNA-binding ANTAR (miR and asR ranscription nti-termination egulators) domain. We show that AvnR plays a role in regulating biofilm formation (on glass and plastic surfaces), surface motility, adhesion to A549 cells as well as in nitrogen metabolism in . RNA-Seq analysis revealed that deletion results in altered expression of more than 150 genes (116 upregulated and 42 downregulated). RNA-Seq data suggest that altered biofilm formation and surface motility observed in the deletion mutant is likely mediated by previously unknown pathways. Of note, was the altered expression of genes predicted to be involved in amino acid transport and metabolism in deletion mutant. Biolog phenotypic array showed that deletion of hampered ATCC17978’s ability to metabolize various nitrogen sources, particularly that of glutamic acid, serine, histidine, aspartic acid, isoleucine and arginine. Taken together our data show that AvnR, the first ANTAR protein described in affects virulence phenotypes as well as its ability to metabolize nitrogen sources.

Funding
This study was supported by the:
  • Ayush kumar , Natural Sciences and Engineering Research Council of Canada , (Award 2015-05550)
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000913
2020-04-23
2020-06-04
Loading full text...

Full text loading...

/deliver/fulltext/micro/10.1099/mic.0.000913/mic000913.html?itemId=/content/journal/micro/10.1099/mic.0.000913&mimeType=html&fmt=ahah

References

  1. Antunes LCS, Visca P, Towner KJ. Acinetobacter baumannii: evolution of a global pathogen. Pathog Dis 2014; 71:292–301 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  2. Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev 2008; 21:538–582 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  3. Wong D, Nielsen TB, Bonomo RA, Pantapalangkoor P, Luna B et al. Clinical and pathophysiological overview of Acinetobacter infections: a century of challenges. Clin Microbiol Rev 2017; 30:409–447 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  4. Dijkshoorn L, Nemec A, Seifert H. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat Rev Microbiol 2007; 5:939–951 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  5. Centers for Disease Control and Prevention Antibiotic resistance threats in the United States Atlanta, GA: U.S: Department of Health and Human Services, CDC; 2019
    [Google Scholar]
  6. World Health Organization Global priority list of antibiotic-resistant bacterial to guide research, discovery, and development of new antibiotics 2017 [Available from: https://www.who.int/medicines/publications/WHO-PPL-Short_Summary_25Feb-ET_NM_WHO.pdf.
  7. Jawad A, Seifert H, Snelling AM, Heritage J, Hawkey PM. Survival of Acinetobacter baumannii on dry surfaces: comparison of outbreak and sporadic isolates. J Clin Microbiol 1998; 36:1938–1941 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  8. Peleg AY, de Breij A, Adams MD, Cerqueira GM, Mocali S et al. The success of Acinetobacter species; genetic, metabolic and virulence attributes. PLoS One 2012; 7:e46984 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  9. Antunes LCS, Imperi F, Carattoli A, Visca P. Deciphering the multifactorial nature of Acinetobacter baumannii pathogenicity. PLoS One 2011; 6:e22674 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  10. Stock AM, Robinson VL, Goudreau PN. Two-component signal transduction. Annu Rev Biochem 2000; 69:183–215 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  11. Jung K, Fried L, Behr S, Heermann R. Histidine kinases and response regulators in networks. Curr Opin Microbiol 2012; 15:118–124 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  12. Mizuno T. His-Asp phosphotransfer signal transduction. J Biochem 1998; 123:555–563 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  13. Alm E, Huang K, Arkin A. The evolution of two-component systems in bacteria reveals different strategies for niche adaptation. PLoS Comput Biol 2006; 2:e143 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  14. Cerqueira GM, Kostoulias X, Khoo C, Aibinu I, Qu Y et al. A global virulence regulator in Acinetobacter baumannii and its control of the phenylacetic acid catabolic pathway. J Infect Dis 2014; 210:46–55 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  15. Tomaras AP, Flagler MJ, Dorsey CW, Gaddy JA, Actis LA. Characterization of a two-component regulatory system from Acinetobacter baumannii that controls biofilm formation and cellular morphology. Microbiology 2008; 154:3398–3409 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  16. Marchand I, Damier-Piolle L, Courvalin P, Lambert T. Expression of the RND-type efflux pump AdeABC in Acinetobacter baumannii is regulated by the AdeRS two-component system. Antimicrob Agents Chemother 2004; 48:3298–3304 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  17. Richmond GE, Evans LP, Anderson MJ, Wand ME, Bonney LC et al. The Acinetobacter baumannii two-component system AdeRS regulates genes required for multidrug efflux, biofilm formation, and virulence in a strain-specific manner. mBio 2016; 7:e00430–16 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  18. Kröger C, Kary SC, Schauer K, Cameron ADS. Genetic regulation of virulence and antibiotic resistance in Acinetobacter baumannii. Genes 2016; 8:12 28 12 2016 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  19. De Silva PM, Kumar A. Signal transduction proteins in Acinetobacter baumannii: role in antibiotic resistance, virulence, and potential as drug targets. Front Microbiol 2019; 10:49 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  20. De Silva PM, Kumar A. Effect of sodium chloride on surface-associated motility of Acinetobacter baumannii and the role of AdeRS two-component system. J Membr Biol 2018; 251:5–13 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  21. Choi K-H, Schweizer HP. An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants. BMC Microbiol 2005; 5:30 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  22. Choi K-H, Gaynor JB, White KG, Lopez C, Bosio CM et al. A Tn7-based broad-range bacterial cloning and expression system. Nat Methods 2005; 2:443–448 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  23. Ducas-Mowchun K, De Silva PM, Patidar R, Schweizer HP, Kumar A. Tn7-based single-copy insertion vectors for Acinetobacter baumannii. In Biswas I, Rather PN. (editors) Acinetobacter baumannii: Methods and Protocols New York, NY: Springer New York; 2019 pp 135–150
    [Google Scholar]
  24. Iwashkiw JA, Seper A, Weber BS, Scott NE, Vinogradov E et al. Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation. PLoS Pathog 2012; 8:e1002758 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  25. Tomaras AP, Dorsey CW, Edelmann RE, Actis LA. Attachment to and biofilm formation on abiotic surfaces by Acinetobacter baumannii: involvement of a novel chaperone-usher pili assembly system. Microbiology 2003; 149:3473–3484 [CrossRef]
    [Google Scholar]
  26. Harding CM, Tracy EN, Carruthers MD, Rather PN, Actis LA et al. Acinetobacter baumannii strain M2 produces type IV pili which play a role in natural transformation and twitching motility but not surface-associated motility. mBio 2013; 4:e00360-13 06 Aug 2013 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  27. Peleg AY, Jara S, Monga D, Eliopoulos GM, Moellering RC et al. Galleria mellonella as a model system to study Acinetobacter baumannii pathogenesis and therapeutics. Antimicrob Agents Chemother 2009; 53:2605–2609 [CrossRef]
    [Google Scholar]
  28. Gaddy JA, Tomaras AP, Actis LA. 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 2009; 77:3150–3160 [CrossRef]
    [Google Scholar]
  29. Álvarez-Fraga L, Pérez A, Rumbo-Feal S, Merino M, Vallejo JA et al. Analysis of the role of the LH92_11085 gene of a biofilm hyper-producing Acinetobacter baumannii strain on biofilm formation and attachment to eukaryotic cells. Virulence 2016; 7:443–455 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  30. Giard DJ, Aaronson SA, Todaro GJ, Arnstein P, Kersey JH et al. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst 1973; 51:1417–1423 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  31. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods 2012; 9:357–359 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  32. Huerta-Cepas J, Szklarczyk D, Forslund K, Cook H, Heller D et al. eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences. Nucleic Acids Res 2016; 44:D286–D293 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  33. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29:e4545 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  34. de Castro E, Sigrist CJA, Gattiker A, Bulliard V, Langendijk-Genevaux PS et al. ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucleic Acids Res 2006; 34:W362–W365 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  35. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 2015; 10:845–858 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  36. Lu S, Wang J, Chitsaz F, Derbyshire MK, Geer RC et al. CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res 2020; 48:D265–D268 [CrossRef]
    [Google Scholar]
  37. Laub MT, Goulian M. Specificity in two-component signal transduction pathways. Annu Rev Genet 2007; 41:121–145 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  38. Casino P, Rubio V, Marina A. Structural insight into partner specificity and phosphoryl transfer in two-component signal transduction. Cell 2009; 139:325–336 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  39. Desai SK, Kenney LJ. To ∼P or not to ∼P? non-canonical activation by two-component response regulators. Mol Microbiol 2017; 103:203–213 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  40. Ramesh A, DebRoy S, Goodson JR, Fox KA, Faz H et al. The mechanism for RNA recognition by ANTAR regulators of gene expression. PLoS Genet 2012; 8:e1002666 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  41. Shu CJ, Zhulin IB. ANTAR: an RNA-binding domain in transcription antitermination regulatory proteins. Trends Biochem Sci 2002; 27:3–5 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  42. Wilson SA, Wachira SJ, Norman RA, Pearl LH, Drew RE. Transcription antitermination regulation of the Pseudomonas aeruginosa amidase operon. Embo J 1996; 15:5907–5916 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  43. Quax TEF, Altegoer F, Rossi F, Li Z, Rodriguez-Franco M et al. Structure and function of the archaeal response regulator CheY. Proc Natl Acad Sci U S A 2018; 115:E1259–E1268 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  44. Liu Y, Lardi M, Pedrioli A, Eberl L, Pessi G. NtrC-dependent control of exopolysaccharide synthesis and motility in Burkholderia cenocepacia H111. PLoS One 2017; 12:e0180362-e [CrossRef][PubMed][PubMed]
    [Google Scholar]
  45. Feirer N, Kim D, Xu J, Fernandez N, Waters CM et al. The Agrobacterium tumefaciens CheY-like protein ClaR regulates biofilm formation. Microbiology 2017; 163:1680–1691 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  46. Cheng AT, Zamorano-Sánchez D, Teschler JK, Wu D, Yildiz FH. NtrC Adds a New Node to the Complex Regulatory Network of Biofilm Formation and vps Expression in Vibrio cholerae. J Bacteriol 2018; 200:e00025–18 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  47. Schröder I, Wolin CD, Cavicchioli R, Gunsalus RP. Phosphorylation and dephosphorylation of the NarQ, NarX, and NarL proteins of the nitrate-dependent two-component regulatory system of Escherichia coli. J Bacteriol 1994; 176:4985–4992 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  48. Tomaras AP, Dorsey CW, Edelmann RE, Actis LA. Attachment to and biofilm formation on abiotic surfaces by Acinetobacter baumannii: involvement of a novel chaperone-usher pili assembly system. Microbiology 2003; 149:3473–3484 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  49. De Silva PM, Chong P, Fernando DM, Westmacott G, Kumar A. Effect of incubation temperature on antibiotic resistance and virulence factors of Acinetobacter baumannii ATCC 17978. Antimicrob Agents Chemother 2018; 62:e01514–01517 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  50. Peleg AY, Jara S, Monga D, Eliopoulos GM, Moellering RC et al. Galleria mellonella as a model system to study Acinetobacter baumannii pathogenesis and therapeutics. Antimicrob Agents Chemother 2009; 53:2605–2609 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  51. Wand ME, Bock LJ, Turton JF, Nugent PG, Sutton JM. Acinetobacter baumannii virulence is enhanced in Galleria mellonella following biofilm adaptation. J Med Microbiol 2012; 61:470–477 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  52. Gaddy JA, Tomaras AP, Actis LA. 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 2009; 77:3150–3160 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  53. Lázaro-Díez M, Navascués-Lejarza T, Remuzgo-Martínez S, Navas J, Icardo JM et al. Acinetobacter baumannii and A. pittii clinical isolates lack adherence and cytotoxicity to lung epithelial cells in vitro. Microbes Infect 2016; 18:559–564 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  54. de Breij A, Gaddy J, van der Meer J, Koning R, Koster A et al. CsuA/BABCDE-dependent pili are not involved in the adherence of Acinetobacter baumannii ATCC19606(T) to human airway epithelial cells and their inflammatory response. Res Microbiol 2009; 160:213–218 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  55. Nait Chabane Y, Marti S, Rihouey C, Alexandre S, Hardouin J et al. Characterisation of pellicles formed by Acinetobacter baumannii at the air-liquid interface. PLoS One 2014; 9:e111660 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  56. Rumbo-Feal S, Gómez MJ, Gayoso C, Álvarez-Fraga L, Cabral MP et al. Whole transcriptome analysis of Acinetobacter baumannii assessed by RNA-sequencing reveals different mRNA expression profiles in biofilm compared to planktonic cells. PLoS One 2013; 8:e72968 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  57. Álvarez-Fraga L, Rumbo-Feal S, Pérez A, Gómez MJ, Gayoso C et al. Global assessment of small RNAs reveals a non-coding transcript involved in biofilm formation and attachment in Acinetobacter baumannii ATCC 17978. PLoS One 2017; 12:e0182084 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  58. Pérez-Varela M, Corral J, Vallejo JA, Rumbo-Feal S, Bou G et al. Mutations in the β-subunit of the RNA polymerase impair the surface-associated motility and virulence of Acinetobacter baumannii. Infect Immun 2017; 85: 19 07 2017 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  59. Mosier AC, Justice NB, Bowen BP, Baran R, Thomas BC et al. Metabolites associated with adaptation of microorganisms to an acidophilic, metal-rich environment identified by stable-isotope-enabled metabolomics. mBio 2013; 4:e00484–12 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  60. Zimmer DP, Soupene E, Lee HL, Wendisch VF, Khodursky AB et al. Nitrogen regulatory protein C-controlled genes of Escherichia coli: scavenging as a defense against nitrogen limitation. Proc Natl Acad Sci U S A 2000; 97:14674–14679 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  61. Ortlund E, Lacount MW, Lewinski K, Lebioda L. Reactions of Pseudomonas 7A glutaminase-asparaginase with diazo analogues of glutamine and asparagine result in unexpected covalent inhibitions and suggests an unusual catalytic triad Thr-Tyr-Glu. Biochemistry 2000; 39:1199–1204 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  62. Swain AL, Jaskólski M, Housset D, Rao JK, Wlodawer A. Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy. Proc Natl Acad Sci U S A 1993; 90:1474–1478 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  63. Cedar H, Schwartz JH. Production of L-asparaginase II by Escherichia coli. J Bacteriol 1968; 96:2043–2048 [CrossRef][PubMed][PubMed]
    [Google Scholar]
  64. Hamad MA, Zajdowicz SL, Holmes RK, Voskuil MI. An allelic exchange system for compliant genetic manipulation of the select agents Burkholderia pseudomallei and Burkholderia mallei. Gene 2009; 430:123–131 [CrossRef][PubMed][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000913
Loading
/content/journal/micro/10.1099/mic.0.000913
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error