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Expression of extracellular polysaccharides and proteins by clinical isolates of Pseudomonas aeruginosa in response to environmental conditions

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Abstract

The opportunistic pathogen Pseudomonas aeruginosa causes chronic respiratory infections in patients with cystic fibrosis (CF). Persistence of this bacterium is attributed to its ability to form biofilms which rely on an extracellular polymeric substance matrix. Extracellular polysaccharides (EPS) and secreted proteins are key matrix components of P. aeruginosa biofilms. Recently, nebulized magnesium sulfate has been reported as a significant bronchodilator for asthmatic patients including CF. However, the impact of magnesium sulfate on the virulence effect of P. aeruginosa is lacking. In this report, we investigated the influence of magnesium sulfate and other environmental factors on the synthesis of alginate and secretion of proteins by a mucoid and a non-mucoid strain of P. aeruginosa, respectively. By applying the Plackett-Burman and Box-Behnken experimental designs, we found that phosphates (6.0 g/l), ammonium sulfate (4.0 g/l), and trace elements (0.6 mg/l) markedly supported alginate production by the mucoid strain. However, ferrous sulfate (0.3 mg/l), magnesium sulfate (0.02 g/l), and phosphates (6.0 g/l) reinforced the secretion of proteins by the non-mucoid strain.

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References

  • Alionte L, Cannon B, White CD, Caballero A, O'Callaghan R, Hobden J (2001) Pseudomonas aeruginosa LasA protease and corneal infections. Curr Eye Res 22:266–271

    Article  PubMed  CAS  Google Scholar 

  • Anwar H, Strap J, Costerton J (1991) Growth characteristics and expression of iron-regulated outer-membrane proteins of chemostat-grown biofilm cells of Pseudomonas aeruginosa. Can J Microbiol 37:737–743

    Article  PubMed  CAS  Google Scholar 

  • Barker AP, Vasil AI, Filloux A, Ball G, Wilderman PJ, Vasil ML (2004) A novel extracellular phospholipase C of Pseudomonas aeruginosa is required for phospholipid chemotaxis. Mol Microbiol 53:1089–1098

    Article  PubMed  CAS  Google Scholar 

  • Baumann U, Wu S, Flaherty KM, McKay DB (1993) Three-dimensional structure of the alkaline protease of Pseudomonas aeruginosa: a two-domain protein with a calcium binding parallel beta roll motif. EMBO J 12:3357

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Bever RA, Iglewski BH (1988) Molecular characterization and nucleotide sequence of the Pseudomonas aeruginosa elastase structural gene. J Bacteriol 170:4309–4314

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Bleves S, Viarre V, Salacha R, Michel GP, Filloux A, Voulhoux R (2010) Protein secretion systems in Pseudomonas aeruginosa: a wealth of pathogenic weapons. Int J Med Microbiol 300:534–543

    Article  PubMed  CAS  Google Scholar 

  • Bonfield TL, Konstan MW, Berger M (1999) Altered respiratory epithelial cell cytokine production in cystic fibrosis. J Allergy Clin Immunol 104:72–78

    Article  PubMed  CAS  Google Scholar 

  • Bothwell MR, Smith AL, Phillips T (2003) Recalcitrant otorrhea due to Pseudomonas biofilm. Otolaryngol Head Neck Surg 129:599–601

    Article  PubMed  Google Scholar 

  • Box GE, Behnken DW (1960) Some new three level designs for the study of quantitative variables. Technometrics 2:455–475

    Article  Google Scholar 

  • Boyd A, Chakrabarty A (1995) Pseudomonas aeruginosa biofilms: role of the alginate exopolysaccharide. J Ind Microbiol Biotechnol 15:162–168

    CAS  Google Scholar 

  • Braun P, de Groot A, Bitter W, Tommassen J (1998) Secretion of elastinolytic enzymes and their propeptides by Pseudomonas aeruginosa. J Bacteriol 180:3467–3469

    PubMed  PubMed Central  CAS  Google Scholar 

  • Clementi F, Moresi M, Parente E (1999) Alginate from Azotobacter vinelandii. In: Bucke C (ed) Carbohydrate biotechnology protocols. Humana Press, Totowa, pp 23–42. https://doi.org/10.1007/978-1-59259-261-6_3

    Chapter  Google Scholar 

  • Döring G, Høiby N (1983) Longitudinal study of immune response to Pseudomonas aeruginosa antigens in cystic fibrosis. Infect Immun 42:197–201

    PubMed  PubMed Central  Google Scholar 

  • Erickson DL, Endersby R, Kirkham A, Stuber K, Vollman DD, Rabin HR, Mitchell I, Storey DG (2002) Pseudomonas aeruginosa quorum-sensing systems may control virulence factor expression in the lungs of patients with cystic fibrosis. Infect Immun 70:1783–1790

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Filloux A (2011) Protein secretion systems in Pseudomonas aeruginosa: an essay on diversity, evolution, and function. Front Microbiol 2:155

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Filloux A, Bally M, Soscia C, Murgier M, Lazdunski A (1988) Phosphate regulation in Pseudomonas aeruginosa: cloning of the alkaline phosphatase gene and identification of phoB-and phoR-like genes. Mol Gen Genet MGG 212:510–513

    Article  PubMed  CAS  Google Scholar 

  • Finck-Barbançon V, Goranson J, Zhu L, Sawa T, Wiener-Kronish JP, Fleiszig SM, Wu C, Mende-Mueller L, Frank DW (1997) ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury. Mol Microbiol 25:547–557

    Article  PubMed  Google Scholar 

  • Fraley CD, Rashid MH, Lee SS, Gottschalk R, Harrison J, Wood PJ, Brown MR, Kornberg A (2007) A polyphosphate kinase 1 (ppk1) mutant of Pseudomonas aeruginosa exhibits multiple ultrastructural and functional defects. Proc Natl Acad Sci 104:3526–3531

    Article  PubMed  CAS  Google Scholar 

  • Franklin MJ, Nivens DE, Weadge JT, Howell PL (2011) Biosynthesis of the Pseudomonas aeruginosa extracellular polysaccharides, alginate, Pel, and Psl. Front Microbiol 2:167

    Article  PubMed  PubMed Central  Google Scholar 

  • Gilbert P, Das J, Foley I (1997) Biofilm susceptibility to antimicrobials. Adv Dent Res 11:160–167

    Article  PubMed  CAS  Google Scholar 

  • Guina T, Wu M, Miller SI, Purvine SO, Eugene CY, Eng J, Goodlett DR, Aebersold R, Ernst RK, Lee KA (2003) Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation. J Am Soc Mass Spectrom 14:742–751

    Article  PubMed  CAS  Google Scholar 

  • Harmsen M, Yang L, Pamp SJ, Tolker-Nielsen T (2010) An update on Pseudomonas aeruginosa biofilm formation, tolerance, and dispersal. FEMS Immunol Med Microbiol 59:253–268

    Article  PubMed  CAS  Google Scholar 

  • Hauser AR, Kang PJ, Engel JN (1998) PepA, a secreted protein of Pseudomonas aeruginosa, is necessary for cytotoxicity and virulence. Mol Microbiol 27:807–818

    Article  PubMed  CAS  Google Scholar 

  • Hay ID, Remminghorst U, Rehm BH (2009) MucR, a novel membrane-associated regulator of alginate biosynthesis in Pseudomonas aeruginosa. Appl Environ Microbiol 75:1110–1120

    Article  PubMed  CAS  Google Scholar 

  • Hinsa SM, Espinosa-Urgel M, Ramos JL, O'toole GA (2003) Transition from reversible to irreversible attachment during biofilm formation by Pseudomonas fluorescens WCS365 requires an ABC transporter and a large secreted protein. Mol Microbiol 49:905–918

    Article  PubMed  CAS  Google Scholar 

  • Hobden JA (2002) Pseudomonas aeruginosa proteases and corneal virulence. DNA Cell Biol 21:391–396

    Article  PubMed  CAS  Google Scholar 

  • Høiby N (1994) Diffuse panbronchiolitis and cystic fibrosis: East meets West. Thorax 49:531–532

    Article  PubMed  PubMed Central  Google Scholar 

  • Høiby N, Ciofu O, Johansen HK, Z-j S, Moser C, Jensen PØ, Molin S, Givskov M, Tolker-Nielsen T, Bjarnsholt T (2011) The clinical impact of bacterial biofilms. Int J Oral Sci 3:55–65

    Article  PubMed  PubMed Central  Google Scholar 

  • Horvat RT, Parmely MJ (1988) Pseudomonas aeruginosa alkaline protease degrades human gamma interferon and inhibits its bioactivity. Infect Immun 56:2925–2932

    PubMed  PubMed Central  CAS  Google Scholar 

  • Keiski C-L, Harwich M, Jain S, Neculai AM, Yip P, Robinson H, Whitney JC, Riley L, Burrows LL, Ohman DE (2010) AlgK is a TPR-containing protein and the periplasmic component of a novel exopolysaccharide secretin. Structure 18:265–273

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Kharazmi A (1991) Mechanisms involved in the evasion of the host defence by Pseudomonas aeruginosa. Immunol Lett 30:201–205

    Article  PubMed  CAS  Google Scholar 

  • Kim K-S, Rao NN, Fraley CD, Kornberg A (2002) Inorganic polyphosphate is essential for long-term survival and virulence factors in Shigella and Salmonella spp. Proc Natl Acad Sci 99:7675–7680

    Article  PubMed  CAS  Google Scholar 

  • Klausen M, Heydorn A, Ragas P, Lambertsen L, Aaes-Jørgensen A, Molin S, Tolker-Nielsen T (2003) Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol Microbiol 48:1511–1524

    Article  PubMed  CAS  Google Scholar 

  • Kornberg A, Rao NN, Ault-Riche D (1999) Inorganic polyphosphate: a molecule of many functions. Annu Rev Biochem 68:89–125

    Article  PubMed  CAS  Google Scholar 

  • Lotfy WA, Abd-El-Karim NM, El-Sharouny EE, El-Helow ER (2017) Isolation and characterization of a haloalkaliphilic protease producer bacterium from Wadi Natrun in Egypt. Afr J Biotechnol 16:1210–1220

    Article  Google Scholar 

  • Lotfy WA, Ghanem KM, Ehab R (2006) Citric acid production by a novel Aspergillus niger isolate: II. Optimization of process parameters through statistical experimental designs. 11: 32–40

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  • Ma L, Wang S, Wang D, Parsek MR, Wozniak DJ (2012) The roles of biofilm matrix polysaccharide Psl in mucoid Pseudomonas aeruginosa biofilms. FEMS Immunol Med Microbiol 65:377–380

    Article  PubMed  CAS  Google Scholar 

  • Martin D, Schurr M, Mudd M, Govan J, Holloway B, Deretic V (1993) Mechanism of conversion to mucoidy in Pseudomonas aeruginosa infecting cystic fibrosis patients. Proc Natl Acad Sci 90:8377–8381

    Article  PubMed  CAS  Google Scholar 

  • Mathee K, Ciofu O, Sternberg C, Lindum PW, Campbell JI, Jensen P, Johnsen AH, Givskov M, Ohman DE, Søren M (1999) Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung. Microbiology 145:1349–1357

    Article  PubMed  CAS  Google Scholar 

  • Matz C, Girskov M (2007) Biofilms as refuge against predation. The Biofilm Mode of Life: Mechanisms and Adaptations, Horizon Scientific Press, pp 195–213

  • McIver KS, Kessler E, Olson JC, Ohman DE (1995) The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa. Mol Microbiol 18:877–889

    Article  PubMed  CAS  Google Scholar 

  • Meluleni GJ, Grout M, Evans DJ, Pier GB (1995) Mucoid Pseudomonas aeruginosa growing in a biofilm in vitro are killed by opsonic antibodies to the mucoid exopolysaccharide capsule but not by antibodies produced during chronic lung infection in cystic fibrosis patients. J Immunol 155:2029–2038

    PubMed  CAS  Google Scholar 

  • Mesaros N, Nordmann P, Plésiat P, Roussel-Delvallez M, Van Eldere J, Glupczynski Y, Van Laethem Y, Jacobs F, Lebecque P, Malfroot A (2007) Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium. Clin Microbiol Infect 13:560–578

    Article  PubMed  CAS  Google Scholar 

  • Meyer J-M (2000) Pyoverdines: pigments, siderophores and potential taxonomic markers of fluorescent Pseudomonas species. Arch Microbiol 174:135–142

    Article  PubMed  CAS  Google Scholar 

  • Mian F, Jarman T, Righelato R (1978) Biosynthesis of exopolysaccharide by Pseudomonas aeruginosa. J Bacteriol 134:418–422

    PubMed  PubMed Central  CAS  Google Scholar 

  • Otterlei M, Østgaard K, Skjåk-Bræk G, Smidsrød O, Soon-Shiong P, Espevik T (1991) Induction of cytokine production from human monocytes stimulated with alginate. J Immunother 10:286–291

    Article  PubMed  CAS  Google Scholar 

  • Phillips RM, Six DA, Dennis EA, Ghosh P (2003) In vivo phospholipase activity of the Pseudomonas aeruginosa cytotoxin ExoU and protection of mammalian cells with phospholipase A2 inhibitors. J Biol Chem 278:41326–41332

    Article  PubMed  CAS  Google Scholar 

  • Plackett RL, Burman JP (1946) The design of optimum multifactorial experiments. Biometrika 33:305–325

    Article  Google Scholar 

  • Purevdorj-Gage B, Costerton W, Stoodley P (2005) Phenotypic differentiation and seeding dispersal in non-mucoid and mucoid Pseudomonas aeruginosa biofilms. Microbiology 151:1569–1576

    Article  PubMed  CAS  Google Scholar 

  • Ramsey DM, Wozniak DJ (2005) Understanding the control of Pseudomonas aeruginosa alginate synthesis and the prospects for management of chronic infections in cystic fibrosis. Mol Microbiol 56:309–322

    Article  PubMed  CAS  Google Scholar 

  • Regni C, Naught L, Tipton PA, Beamer LJ (2004) Structural basis of diverse substrate recognition by the enzyme PMM/PGM from P. aeruginosa. Structure 12:55–63

    Article  PubMed  CAS  Google Scholar 

  • Regni C, Tipton PA, Beamer LJ (2002) Crystal structure of PMM/PGM: an enzyme in the biosynthetic pathway of P. aeruginosa virulence factors. Structure 10:269–279

    Article  PubMed  CAS  Google Scholar 

  • Römling U, Schmidt KD, Tümmler B (1997) Large genome rearrangements discovered by the detailed analysis of 21 Pseudomonas aeruginosa clone C isolates found in environment and disease habitats. J Mol Biol 271:386–404

    Article  PubMed  Google Scholar 

  • Sarhan HA, El-Garhy OH, Ali MA, Youssef NA (2016) The efficacy of nebulized magnesium sulfate alone and in combination with salbutamol in acute asthma. Drug Des Devel Ther 10:1927

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Schlictman D, Kavanaugh-Black A, Shankar S, Chakrabarty A (1994) Energy metabolism and alginate biosynthesis in Pseudomonas aeruginosa: role of the tricarboxylic acid cycle. J Bacteriol 176:6023–6029

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Schmidt KD, Tümmler B, Römling U (1996) Comparative genome mapping of Pseudomonas aeruginosa PAO with P. aeruginosa C, which belongs to a major clone in cystic fibrosis patients and aquatic habitats. J Bacteriol 178:85–93

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Singh G, Tamboli E, Acharya A, Kumarasamy C, Mala K, Raman P (2015) Bioactive proteins from Solanaceae as quorum sensing inhibitors against virulence in Pseudomonas aeruginosa. Med Hypotheses 84:539–542

    Article  PubMed  CAS  Google Scholar 

  • Tielen P, Kuhn H, Rosenau F, Jaeger K-E, Flemming H-C, Wingender J (2013) Interaction between extracellular lipase LipA and the polysaccharide alginate of Pseudomonas aeruginosa. BMC Microbiol 13:159

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Tielen P, Rosenau F, Wilhelm S, Jaeger K-E, Flemming H-C, Wingender J (2010) Extracellular enzymes affect biofilm formation of mucoid Pseudomonas aeruginosa. Microbiology 156:2239–2252

    Article  PubMed  CAS  Google Scholar 

  • Toyofuku M, Roschitzki B, Riedel K, Eberl L (2012) Identification of proteins associated with the Pseudomonas aeruginosa biofilm extracellular matrix. J Proteome Res 11:4906–4915

    Article  PubMed  CAS  Google Scholar 

  • van ‘t Wout EF, van Schadewijk A, van Boxtel R, Dalton LE, Clarke HJ, Tommassen J, Marciniak SJ, Hiemstra PS (2015) Virulence factors of Pseudomonas aeruginosa induce both the unfolded protein and integrated stress responses in airway epithelial cells. PLoS Pathog 11:e1004946

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Voulhoux R, Ball G, Ize B, Vasil ML, Lazdunski A, Wu LF, Filloux A (2001) Involvement of the twin-arginine translocation system in protein secretion via the type II pathway. EMBO J 20:6735–6741

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Voulhoux R, Bos MP, Geurtsen J, Mols M, Tommassen J (2003) Role of a highly conserved bacterial protein in outer membrane protein assembly. Science 299:262–265

    Article  PubMed  CAS  Google Scholar 

  • Wang S, Hao Y, Lam JS, Vlahakis JZ, Szarek WA, Vinnikova A, Veselovsky VV, Brockhausen I (2015) Biosynthesis of the common polysaccharide antigen of Pseudomonas aeruginosa PAO1: characterization and role of GDP-d-rhamnose: GlcNAc/GalNAc-diphosphate-lipid α1, 3-d-rhamnosyltransferase WbpZ. J Bacteriol 197:2012–2019

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Whitney JC, Hay ID, Li C, Eckford PD, Robinson H, Amaya MF, Wood LF, Ohman DE, Bear CE, Rehm BH (2011) Structural basis for alginate secretion across the bacterial outer membrane. Proc Natl Acad Sci 108:13083–13088

    Article  PubMed  Google Scholar 

  • Xiao G, Déziel E, He J, Lépine F, Lesic B, Castonguay MH, Milot S, Tampakaki AP, Stachel SE, Rahme LG (2006) MvfR, a key Pseudomonas aeruginosa pathogenicity LTTR-class regulatory protein, has dual ligands. Mol Microbiol 62:1689–1699

    Article  PubMed  CAS  Google Scholar 

  • Yahr TL, Goranson J, Frank DW (1996) Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway. Mol Microbiol 22:991–1003

    Article  PubMed  CAS  Google Scholar 

  • Yahr TL, Wolfgang MC (2006) Transcriptional regulation of the Pseudomonas aeruginosa type III secretion system. Mol Microbiol 62:631–640

    Article  PubMed  CAS  Google Scholar 

  • Yang L, Hu Y, Liu Y, Zhang J, Ulstrup J, Molin S (2011) Distinct roles of extracellular polymeric substances in Pseudomonas aeruginosa biofilm development. Environ Microbiol 13:1705–1717

    Article  PubMed  CAS  Google Scholar 

  • Yang X, Kuk J, Moffat K (2008) Crystal structure of Pseudomonas aeruginosa bacteriophytochrome: photoconversion and signal transduction. Proc Natl Acad Sci 105:14715–14720

    Article  PubMed  Google Scholar 

  • Yu X, Hallett S, Sheppard J, Watson A (1997) Application of the Plackett-Burman experimental design to evaluate nutritional requirements for the production of Colletotrichum coccodes spores. Appl Microbiol Biotechnol 47:301–305

    Article  CAS  Google Scholar 

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Correspondence to Walid A. Lotfy.

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Lotfy, W.A., Atalla, R.G., Sabra, W.A. et al. Expression of extracellular polysaccharides and proteins by clinical isolates of Pseudomonas aeruginosa in response to environmental conditions. Int Microbiol 21, 129–142 (2018). https://doi.org/10.1007/s10123-018-0010-5

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