Expression of mucoid induction factor MucE is dependent upon the alternate sigma factor AlgU in Pseudomonas aeruginosa

BMC Microbiology

Hongwei D Yu¹  Michael J Schurr⁶  Xin Wang⁵  Christopher L Pritchett⁴  T Ryan Withers²  F Heath Damron³  Yeshi Yin⁵ 

[1] Progenesis Technologies, LLC, 1111 Veterans Memorial Blvd, Huntington, WV 25701, USA;Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine at Marshall University, Huntington, WV 25755, USA;Division of Infectious Diseases and International Health, University of Virginia, Box 800419, MR-6, Charlottesville, VA 22908, USA;Department of Health Sciences, East Tennessee State University, Johnson City, TN 37615, USA;Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, No. 198, Shiqiao Road, Hangzhou 310021, China;Department of Microbiology, School of Medicine, University of Colorado, Aurora, Colorado 80045, USA

关键词: Sigma factor;    MucE;    AlgU/T;    Mucoidy;    Alginate;    Pseudomonas aeruginosa;   

Others  :  1142828
DOI  :  10.1186/1471-2180-13-232

 received in 2013-05-29, accepted in 2013-10-09,  发布年份 2013

【 摘 要 】

Background

Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T (σ²²) to initiate transcription of the alginate biosynthetic operon.

Results

In the current study, we mapped the mucE transcriptional start site, and determined that P_mucE activity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in P_mucE activity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ΔalgU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE.

Conclusions

Our data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa.

【 授权许可】

   
2013 Yin et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Govan JR, Deretic V: Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 1996, 60(3):539-574.
• [2]May TB, Shinabarger D, Maharaj R, Kato J, Chu L, DeVault JD, Roychoudhury S, Zielinski NA, Berry A, Rothmel RK, et al.: Alginate synthesis by Pseudomonas aeruginosa: a key pathogenic factor in chronic pulmonary infections of cystic fibrosis patients. Clin Microbiol Rev 1991, 4(2):191-206.
• [3]Leid JG, Willson CJ, Shirtliff ME, Hassett DJ, Parsek MR, Jeffers AK: The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing. J Immunol 2005, 175(11):7512-7518.
• [4]Pier GB, Coleman F, Grout M, Franklin M, Ohman DE: Role of alginate O acetylation in resistance of mucoid Pseudomonas aeruginosa to opsonic phagocytosis. Infect Immun 2001, 69(3):1895-1901.
• [5]Martin DW, Holloway BW, Deretic V: Characterization of a locus determining the mucoid status of Pseudomonas aeruginosa: AlgU shows sequence similarities with a Bacillus sigma factor. J Bacteriol 1993, 175(4):1153-1164.
• [6]Hershberger CD, Ye RW, Parsek MR, Xie ZD, Chakrabarty AM: The algT (algU) gene of Pseudomonas aeruginosa, a key regulator involved in alginate biosynthesis, encodes an alternative sigma factor (sigma E). Proc Natl Acad Sci U S A 1995, 92(17):7941-7945.
• [7]Xie ZD, Hershberger CD, Shankar S, Ye RW, Chakrabarty AM: Sigma factor-anti-sigma factor interaction in alginate synthesis: inhibition of AlgT by MucA. J Bacteriol 1996, 178(16):4990-4996.
• [8]Damron FH, Goldberg JB: Proteolytic regulation of alginate overproduction in Pseudomonas aeruginosa. Mol Microbiol 2012, 84(4):595-607.
• [9]Qiu D, Eisinger VM, Rowen DW, Yu HD: Regulated proteolysis controls mucoid conversion in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 2007, 104(19):8107-8112.
• [10]Lizewski SE, Schurr JR, Jackson DW, Frisk A, Carterson AJ, Schurr MJ: Identification of AlgR-regulated genes in Pseudomonas aeruginosa by use of microarray analysis. J Bacteriol 2004, 186(17):5672-5684.
• [11]Figurski DH, Helinski DR: Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 1979, 76(4):1648-1652.
• [12]Hoang TT, Kutchma AJ, Becher A, Schweizer HP: Integration-proficient plasmids for Pseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains. Plasmid 2000, 43(1):59-72.
• [13]Miller JH: Beta-Galactosidase Assay. In Experiments in Molecular Genetics. Edited by Miller JH. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory; 1972:352-355.
• [14]Damron FH, Qiu D, Yu HD: The Pseudomonas aeruginosa sensor kinase KinB negatively controls alginate production through AlgW-dependent MucA proteolysis. J Bacteriol 2009, 191(7):2285-2295.
• [15]Damron FH, Owings JP, Okkotsu Y, Varga JJ, Schurr JR, Goldberg JB, Schurr MJ, Yu HD: Analysis of the Pseudomonas aeruginosa regulon controlled by the sensor kinase KinB and sigma factor RpoN. J Bacteriol 2012, 194(6):1317-1330.
• [16]Qiu D, Damron FH, Mima T, Schweizer HP, Yu HD: PBAD-based shuttle vectors for functional analysis of toxic and highly regulated genes in Pseudomonas and Burkholderia spp. and other bacteria. Appl Environ Microbiol 2008, 74(23):7422-7742.
• [17]Wood LF, Ohman DE: Use of cell wall stress to characterize sigma 22 (AlgT/U) activation by regulated proteolysis and its regulon in Pseudomonas aeruginosa. Mol Microbiol 2009, 72(1):183-201.
• [18]Damron FH, Davis MR Jr, Withers TR, Ernst RK, Goldberg JB, Yu G, Yu HD: Vanadate and triclosan synergistically induce alginate production by Pseudomonas aeruginosa strain PAO1. Mol Microbiol 2011, 81(2):554-570.
• [19]Boucher JC, Schurr MJ, Deretic V: Dual regulation of mucoidy in Pseudomonas aeruginosa and sigma factor antagonism. Mol Microbiol 2000, 36(2):341-351.
• [20]Suh SJ, Silo-Suh L, Woods DE, Hassett DJ, West SE, Ohman DE: Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa. J Bacteriol 1999, 181(13):3890-3897.
• [21]Schurr MJ, Martin DW, Mudd MH, Deretic V: Gene cluster controlling conversion to alginate-overproducing phenotype in Pseudomonas aeruginosa: functional analysis in a heterologous host and role in the instability of mucoidy. J Bacteriol 1994, 176(11):3375-3382.
• [22]DeVries CA, Ohman DE: Mucoid-to-nonmucoid conversion in alginate-producing Pseudomonas aeruginosa often results from spontaneous mutations in algT, encoding a putative alternate sigma factor, and shows evidence for autoregulation. J Bacteriol 1994, 176(21):6677-6687.
• [23]Damkiaer S, Yang L, Molin S, Jelsbak L: Evolutionary remodeling of global regulatory networks during long-term bacterial adaptation to human hosts. Proc Natl Acad Sci U S A 2013, 110(19):7766-7771.
• [24]Yeshi Y, Ryan Withers T, Xin W, Yu HD: Evidence for sigma factor competition in the regulation of alginate production by Pseudomonas aeruginosa. PLoS ONE 2013, 8(8):e72329.
• [25]Martin DW, Schurr MJ, Yu H, Deretic V: Analysis of promoters controlled by the putative sigma factor AlgU regulating conversion to mucoidy in Pseudomonas aeruginosa: relationship to sigma E and stress response. J Bacteriol 1994, 176(21):6688-6696.
• [26]Firoved AM, Boucher JC, Deretic V: Global genomic analysis of AlgU (sigma(E))-dependent promoters (sigmulon) in Pseudomonas aeruginosa and implications for inflammatory processes in cystic fibrosis. J Bacteriol 2002, 184(4):1057-1064.
• [27]Wood LF, Leech AJ, Ohman DE: Cell wall-inhibitory antibiotics activate the alginate biosynthesis operon in Pseudomonas aeruginosa: Roles of sigma (AlgT) and the AlgW and Prc proteases. Mol Microbiol 2006, 62(2):412-426.
• [28]Wurtzel O, Yoder-Himes DR, Han K, Dandekar AA, Edelheit S, Greenberg EP, Sorek R, Lory S: The single-nucleotide resolution transcriptome of Pseudomonas aeruginosa grown in body temperature. PLoS Pathog 2012, 8(9):e1002945.
• [29]Damron FH, Napper J, Teter MA, Yu HD: Lipotoxin F of Pseudomonas aeruginosa is an AlgU-dependent and alginate-independent outer membrane protein involved in resistance to oxidative stress and adhesion to A549 human lung epithelia. Microbiology 2009, 155(Pt 4):1028-1038.
• [30]Boucher JC, Yu H, Mudd MH, Deretic V: Mucoid Pseudomonas aeruginosa in cystic fibrosis: characterization of muc mutations in clinical isolates and analysis of clearance in a mouse model of respiratory infection. Infect Immun 1997, 65(9):3838-3846.
• [31]Qiu D, Eisinger VM, Head NE, Pier GB, Yu HD: ClpXP proteases positively regulate alginate overexpression and mucoid conversion in Pseudomonas aeruginosa. Microbiology 2008, 154(Pt 7):2119-2130.
• [32]Cezairliyan BO, Sauer RT: Control of Pseudomonas aeruginosa AlgW protease cleavage of MucA by peptide signals and MucB. Mol Microbiol 2009, 72(2):368-379.
• [33]Garrett ES, Perlegas D, Wozniak DJ: Negative control of flagellum synthesis in Pseudomonas aeruginosa is modulated by the alternative sigma factor AlgT (AlgU). J Bacteriol 1999, 181(23):7401-7404.
• [34]Diggle SP, Winzer K, Lazdunski A, Williams P, Camara 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.
• [35]Caldas TD, El Yaagoubi A, Richarme G: Chaperone properties of bacterial elongation factor EF-Tu. J Biol Chem 1998, 273(19):11478-11482.
• [36]Barbier M, Owings JP, Martinez-Ramos I, Damron FH, Gomila R, Blazquez J, Goldberg JB, Alberti S: Lysine trimethylation of EF-Tu mimics platelet-activating factor to initiate Pseudomonas aeruginosa pneumonia. MBio 2013, 4(3):e00207-e00213.
• [37]Barbier M, Oliver A, Rao J, Hanna SL, Goldberg JB, Alberti S: Novel phosphorylcholine-containing protein of Pseudomonas aeruginosa chronic infection isolates interacts with airway epithelial cells. J Infect Dis 2008, 197(3):465-473.
• [38]Yu H, Boucher JC, Hibler NS, Deretic V: Virulence properties of Pseudomonas aeruginosa lacking the extreme-stress sigma factor AlgU (sigmaE). Infect Immun 1996, 64(7):2774-2781.