The Cyclic AMP-Binding Protein CbpB in Brucella melitensis and its role in cell envelope integrity, resistance to detergent and virulence
Wen-juan Liu
Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China
Search for more papers by this authorHao Dong
Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China
Search for more papers by this authorXiao-wei Peng
Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China
Search for more papers by this authorCorresponding Author
Qing-min Wu
Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China
Correspondence: Qingmin Wu, Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. Tel./Fax: +86 10 6273 3901; e-mail: [email protected]Search for more papers by this authorWen-juan Liu
Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China
Search for more papers by this authorHao Dong
Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China
Search for more papers by this authorXiao-wei Peng
Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China
Search for more papers by this authorCorresponding Author
Qing-min Wu
Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China
Correspondence: Qingmin Wu, Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. Tel./Fax: +86 10 6273 3901; e-mail: [email protected]Search for more papers by this authorAbstract
Brucella melitensis possesses an operon with two components: the response regulator OtpR and a putative cAMP-dependent protein kinase regulatory subunit encoded by the BMEI0067 gene. In the previous study, the function of OtpR has been studied, while little is known about the function of the BMEI0067 gene. Using a bioinformatics approach, we showed that the BMEI0067 gene encodes an additional putative cAMP-binding protein, which we refer to as CbpB. Structural modeling predicted that CbpB has a cAMP-binding protein (CAP) domain and is structurally similar to eukaryotic protein kinase A regulatory subunits. Here, we report the characterization of CbpB, a cAMP-binding protein in Brucella melitensis, showed to be involved in mouse persistent infections. ∆cbpB::km possessed cell elongation, bubble-like protrusions on cell surface and its resistance to environmental stresses (temperature, osmotic stress and detergent). Interestingly, comparative real-time qPCR assays, the cbpB mutation resulted in significantly different expression of aqpX and several penicillin-binding proteins and cell division proteins in Brucella. Combined, these results demonstrated characterization of CbpB in B. melitensis and its key role for intracellular multiplication.
Supporting Information
Filename | Description |
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fml12472-sup-0001-FigS1-2-TableS1.pdfapplication/PDF, 178.4 KB | Table S1. Primers used in this work. Fig. S1. Alignment of the CAP domains. Fig. S2. Growth of B. melitensis cpK mutant in low K+. |
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References
- Altarejos JY & Montminy M (2011) CREB and the CRTC co-activators: sensors for hormonal and metabolic signals. Nat Rev Mol Cell Biol 12: 141–151.
- Alton GG, Jones LM & Pietz DE (1975) Laboratory techniques in brucellosis. Monogr Ser World Health Organ 55: 1–163.
- Banai M, Adams LG, Frey M, Pugh R & Ficht TA (2002) The myth of Brucella L-forms and possible involvement of Brucella penicillin binding proteins (PBPs) in pathogenicity. Vet Microbiol 90: 263–279.
- Bandara AB, Schurig GG, Sriranganathan N, Prasad R & Boyle SM (2009) The putative penicillin-binding proteins 1 and 2 are important for viability, growth and cell morphology of Brucella melitensis. Vet Microbiol 133: 387–393.
- Bisicchia P, Arumugam S, Schwille P & Sherratt D (2013) MinC, MinD, and MinE drive counter-oscillation of early-cell-division proteins prior to Escherichia coli septum formation. MBio 4: e813–e856.
- Boschiroli ML, Foulongne V & O'Callaghan D (2001) Brucellosis: a worldwide zoonosis. Curr Opin Microbiol 4: 58–64.
- Botsford JL & Harman JG (1992) Cyclic AMP in prokaryotes. Microbiol Rev 56: 100–122.
- Celli J, de Chastellier C, Franchini DM, Pizarro-Cerda J, Moreno E & Gorvel JP (2003) Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum. J Exp Med 198: 545–556.
- Clegg CH, Cadd GG & McKnight GS (1988) Genetic characterization of a brain-specific form of the type I regulatory subunit of cAMP-dependent protein kinase. P Natl Acad Sci USA 85: 3703–3707.
- Delamarche C, Thomas D, Rolland JP et al. (1999) Visualization of AqpZ-mediated water permeability in Escherichia coli by cryoelectron microscopy. J Bacteriol 181: 4193–4197.
- Diaz R, Jones LM & Wilson JB (1968) Antigenic relationship of the gram-negative organism causing canine abortion to smooth and rough brucellae. J Bacteriol 95: 618–624.
- Endoh T & Engel JN (2009) CbpA: a polarly localized novel cyclic AMP-binding protein in Pseudomonas aeruginosa. J Bacteriol 191: 7193–7205.
- Erdogan S, Celik S, Aslantas O, Kontas T & Ocak S (2007) Elevated cAMP levels reverse Brucella melitensis-induced lipid peroxidation and stimulate IL-10 transcription in rats. Res Vet Sci 82: 181–186.
- Erdogan S, Aslantas O, Celik S & Atik E (2008) The effects of increased cAMP content on inflammation, oxidative stress and PDE4 transcripts during Brucella melitensis infection. Res Vet Sci 84: 18–25.
- Hernandez-Castro R, Rodriguez MC, Seoane A & Garcia LJ (2003) The aquaporin gene aqpX of Brucella abortus is induced in hyperosmotic conditions. Microbiology 149: 3185–3192.
- Kaneko T, Nakamura Y, Sato S et al. (2000) Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti. DNA Res 7: 331–338.
- Khankal R, Chin JW, Ghosh D & Cirino PC (2009) Transcriptional effects of CRP* expression in Escherichia coli. J Biol Eng 3: 13.
- Kimura Y, Nakato H, Ishibashi K & Kobayashi S (2005) A Myxococcus xanthus CbpB containing two cAMP-binding domains is involved in temperature and osmotic tolerances. FEMS Microbiol Lett 244: 75–83.
- Kimura Y, Kakemizu A, Matsubara Y & Takegawa K (2009) Interaction between a Ser/Thr protein kinase, SpkA, and a cAMP-dependent protein kinase regulatory subunit homolog, CbpB, from Myxococcus xanthus. J Gen Appl Microbiol 55: 499–502.
- Kolb A, Busby S, Buc H, Garges S & Adhya S (1993) Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem 62: 749–795.
- Liu W, Dong H, Liu W, Gao X, Zhang C & Wu Q (2012) OtpR regulated the growth, cell morphology of B. melitensis and tolerance to beta-lactam agents. Vet Microbiol 159: 90–98.
- Marchesini MI, Herrmann CK, Salcedo SP, Gorvel JP & Comerci DJ (2011) In search of Brucella abortus type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system. Cell Microbiol 13: 1261–1274.
- Mari SA, Pessoa J, Altieri S, Hensen U, Thomas L, Morais-Cabral JH & Muller DJ (2011) Gating of the MlotiK1 potassium channel involves large rearrangements of the cyclic nucleotide-binding domains. P Natl Acad Sci USA 108: 20802–20807.
- McDonough KA & Rodriguez A (2012) The myriad roles of cyclic AMP in microbial pathogens: from signal to sword. Nat Rev Microbiol 10: 27–38.
- Mirabella A, Terwagne M, Zygmunt MS, Cloeckaert A, De Bolle X & Letesson JJ (2013) Brucella melitensis MucR, an orthologue of Sinorhizobium meliloti MucR, is involved in resistance to oxidative, detergent, and saline stresses and cell envelope modifications. J Bacteriol 195: 453–465.
- Pei J, Turse JE, Wu Q & Ficht TA (2006) Brucella abortus rough mutants induce macrophage oncosis that requires bacterial protein synthesis and direct interaction with the macrophage. Infect Immun 74: 2667–2675.
- Rickman L, Scott C, Hunt DM et al. (2005) A member of the cAMP receptor protein family of transcription regulators in Mycobacterium tuberculosis is required for virulence in mice and controls transcription of the rpfA gene coding for a resuscitation promoting factor. Mol Microbiol 56: 1274–1286.
- Robertson GT & Roop RJ (1999) The Brucella abortus host factor I (HF-I) protein contributes to stress resistance during stationary phase and is a major determinant of virulence in mice. Mol Microbiol 34: 690–700.
- Schunke S, Stoldt M, Novak K, Kaupp UB & Willbold D (2009) Solution structure of the Mesorhizobium loti K1 channel cyclic nucleotide-binding domain in complex with cAMP. EMBO Rep 10: 729–735.
- Schurig GG, Roop RN, Bagchi T, Boyle S, Buhrman D & Sriranganathan N (1991) Biological properties of RB51; a stable rough strain of Brucella abortus. Vet Microbiol 28: 171–188.
- Seitz LC & Brun YV (1998) Genetic analysis of mecillinam-resistant mutants of Caulobacter crescentus deficient in stalk biosynthesis. J Bacteriol 180: 5235–5239.
- Zhang X, Ren J, Li N, Liu W & Wu Q (2009) Disruption of the BMEI0066 gene attenuates the virulence of Brucella melitensis and decreases its stress tolerance. Int J Biol Sci 5: 570–577.