Autolytic Activity and Plasma Binding Study of Aap, a Novel Minor Autolysin of Streptococcus pneumoniae
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Abstract
Pneumococcal autolysins are enzymes involved in cell wall turnover and cellular division physiologically. They have been found to be involved in the pneumococcus pathogenesis. The aim of this study was to identify the autolytic activity of Spr1754 as a novel protein of Streptococcus pneumoniae. Moreover, the binding of the recombinant protein to plasma proteins was also determined. The spr1754 gene was amplified by PCR and cloned into the pET21a(+) prokaryotic expression vector. The constructed pET21a(+)/spr1754 recombinant plasmid was transformed into E. coli Origami (DE3) and induced using IPTG. The recombinant protein of Spr1754 was purified by Ni-NTA affinity chromatography and confirmed by SDS-PAGE and Western blot analysis using anti-His tag monoclonal antibody. Autolytic activity and the ability of the recombinant protein in binding to plasma proteins were performed using zymogram analysis and western blot, respectively. The spr1754 with expected size was cloned and overexpressed in Escherichia coli Origami (DE3), successfully. After purification of the Spr1754 recombinant protein, the autolytic activity was observed by zymography. Of the four plasma proteins used in this study, binding of lactoferrin to Spr1754 recombinant protein was shown. The Spr1754 recombinant protein has a bifunctional activity, i.e., as being autolysin and lactoferrin binding and designated as Aap (autolytic/ adhesion/ pneumococcus). Nevertheless, characterization of the Aap needs to be followed using gene inactivation and cell wall localization.
(1) Domingues S, Aires AC, Mohedano ML, Lopez P, Arraiano CM. A new tool for cloning and gene expression in Streptococcus pneumoniae. Plasmid 2013 Sep;70(2):247-53.
(2) Ng EW, Costa JR, Samiy N, Ruoff KL, Connolly E, Cousins FV, et al. Contribution of pneumolysin and autolysin to the pathogenesis of experimental pneumococcal endophthalmitis. Retina 2002 Oct;22(5):622-32.
(3) Hirst RA, Gosai B, Rutman A, Guerin CJ, Nicotera P, Andrew PW, et al. Streptococcus pneumoniae deficient in pneumolysin or autolysin has reduced virulence in meningitis. J Infect Dis 2008 Mar 1;197(5):744-51.
(4) Tuomanen E, Liu H, Hengstler B, Zak O, Tomasz A. The induction of meningeal inflammation by components of the pneumococcal cell wall. J Infect Dis 1985 May;151(5):859-68.
(5) Whatmore AM, Dowson CG. The autolysin-encoding gene (lytA) of Streptococcus pneumoniae displays restricted allelic variation despite localized recombination events with genes of pneumococcal bacteriophage encoding cell wall lytic enzymes. Infect Immun 1999 Sep;67(9):4551-6.
(6) Ju CX, Gu HW, Lu CP. Characterization and functional analysis of atl, a novel gene encoding autolysin in Streptococcus suis. J Bacteriol 2012 Mar;194(6):1464-73.
(7) Ingavale SS, van WW, Cheung AL. Characterization of RAT, an autolysis regulator in Staphylococcus aureus. Mol Microbiol 2003 Jun;48(6):1451-66.
(8) Ramadurai L, Lockwood KJ, Nadakavukaren MJ, Jayaswal RK. Characterization of a chromosomally encoded glycylglycine endopeptidase of Staphylococcus aureus. Microbiology 1999 Apr;145 ( Pt 4):801-8.
(9) Ramadurai L, Jayaswal RK. Molecular cloning, sequencing, and expression of lytM, a unique autolytic gene of Staphylococcus aureus. J Bacteriol 1997 Jun;179(11):3625-31.
(10) Diaz E, Lopez R, Garcia JL. Chimeric phage-bacterial enzymes: a clue to the modular evolution of genes. Proc Natl Acad Sci U S A 1990 Oct;87(20):8125-9.
(11) Mellroth P, Daniels R, Eberhardt A, Ronnlund D, Blom H, Widengren J, et al. LytA, major autolysin of Streptococcus pneumoniae, requires access to nascent peptidoglycan. J Biol Chem 2012 Mar 30;287(14):11018-29.
(12) Greve T, Moller JK. Accuracy of using the lytA gene to distinguish Streptococcus pneumoniae from related species. J Med Microbiol 2012 Apr;61(Pt 4):478-82.
(13) Abdeldaim G, Herrmann B, Molling P, Holmberg H, Blomberg J, Olcen P, et al. Usefulness of real-time PCR for lytA, ply, and Spn9802 on plasma samples for the diagnosis of pneumococcal pneumonia. Clin Microbiol Infect 2010 Aug;16(8):1135-41.
(14) Jung CJ, Zheng QH, Shieh YH, Lin CS, Chia JS. Streptococcus mutans autolysin AtlA is a fibronectin-binding protein and contributes to bacterial survival in the bloodstream and virulence for infective endocarditis. Mol Microbiol 2009 Nov;74(4):888-902.
(15) Bourgeois I, Camiade E, Biswas R, Courtin P, Gibert L, Gotz F, et al. Characterization of AtlL, a bifunctional autolysin of Staphylococcus lugdunensis with N-acetylglucosaminidase and N-acetylmuramoyl-l-alanine amidase activities. FEMS Microbiol Lett 2009 Jan;290(1):105-13.
(16) Kurien BT, Scofield RH. A brief review of other notable protein detection methods on acrylamide gels. Methods Mol Biol 2012;869:617-20.
(17) Mahmood T, Yang PC. Western blot: technique, theory, and trouble shooting. N Am J Med Sci 2012 Sep;4(9):429-34.
(18) Ju CX, Gu HW, Lu CP. Characterization and functional analysis of atl, a novel gene encoding autolysin in Streptococcus suis. J Bacteriol 2012 Mar;194(6):1464-73.
(19) Hakansson A, Roche H, Mirza S, McDaniel LS, Brooks-Walter A, Briles DE. Characterization of binding of human lactoferrin to pneumococcal surface protein A. Infect Immun 2001 May;69(5):3372-81.
(20) Chia JS, Yeh CY, Chen JY. Identification of a fibronectin binding protein from Streptococcus mutans. Infect Immun 2000 Apr;68(4):1864-70.
(21) Hammerschmidt S, Bethe G, Remane PH, Chhatwal GS. Identification of pneumococcal surface protein A as a lactoferrin-binding protein of Streptococcus pneumoniae. Infect Immun 1999 Apr;67(4):1683-7.
(22) Shockman GD, Daneo-Moore L, Kariyama R, Massidda O. Bacterial walls, peptidoglycan hydrolases, autolysins, and autolysis. Microb Drug Resist 1996;2(1):95-8.
(23) Foster SJ. The role and regulation of cell wall structural dynamics during differentiation of endospore-forming bacteria. Soc Appl Bacteriol Symp Ser 1994;23:25S-39S.
(24) Heilmann C, Hartleib J, Hussain MS, Peters G. The multifunctional Staphylococcus aureus autolysin aaa mediates adherence to immobilized fibrinogen and fibronectin. Infect Immun 2005 Aug;73(8):4793-802.
(25) Sourav S, Patricia A, Sharma S, Kanungo R, Jayachandran S, Prashanth K. Detection of pneumolysin and autolysin genes among antibiotic resistant Streptococcus pneumoniae in invasive infections. Indian J Med Microbiol 2010 Jan;28(1):34-9.
(26) McLaughlan AM, Foster SJ. Molecular characterization of an autolytic amidase of Listeria monocytogenes EGD. Microbiology 1998 May;144 ( Pt 5):1359-67.
(27) Garcia E, Garcia JL, Garcia P, Arraras A, Sanchez-Puelles JM, Lopez R. Molecular evolution of lytic enzymes of Streptococcus pneumoniae and its bacteriophages. Proc Natl Acad Sci U S A 1988 Feb;85(3):914-8.
(28) Lorente ML, Falguera M, Nogues A, Gonzalez AR, Merino MT, Caballero MR. Diagnosis of pneumococcal pneumonia by polymerase chain reaction (PCR) in whole blood: a prospective clinical study. Thorax 2000 Feb;55(2):133-7.
(29) Sheppard CL, Harrison TG, Morris R, Hogan A, George RC. Autolysin-targeted LightCycler assay including internal process control for detection of Streptococcus pneumoniae DNA in clinical samples. J Med Microbiol 2004 Mar;53(Pt 3):189-95.
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| Issue | Vol 54, No 3 (2016) | |
| Section | Original Articles | |
| Keywords | ||
| Streptococcus pneumonia Minor autolysin Spr 1754 | ||
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