Identification of minor inner-membrane components of the Shigella type III secretion system 'needle complex'.
Type III secretion systems (T3SSs or secretons) are central virulence factors of many Gram-negative bacteria, used to inject protein effectors of virulence into eukaryotic host cells. Their overall morphology, consisting of a cytoplasmic region, an inner- and outer-membrane section and an extracellular needle, is conserved in various species. A portion of the secreton, containing the transmembrane regions and needle, has been isolated biochemically and termed the 'needle complex' (NC). However, there are still unsolved questions concerning the nature and relative arrangement of the proteins assembling the NC. Until these are resolved, the mode of function of the NC cannot be clarified. This paper describes an affinity purification method that enables highly efficient purification of Shigella NCs under near-physiological conditions. Using this method, three new minor components of the NC were identified by mass spectrometry: IpaD, a known component of the needle tip complex, and two predicted components of its central inner-membrane export apparatus, Spa40 and Spa24. A further minor component of the NC, MxiM, is only detected by immunoblotting. MxiM is a 'pilotin'-type protein for the outer-membrane 'secretin' ring formed of MxiD. As expected, it localized to the outer rim of the upper ring of NCs, validating the other findings.
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
Sebastian F
SF
Zenk
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
David
D
Stabat
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
Julie L
JL
Hodgkinson
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
Andreas K J
AKJ
Veenendaal
Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.|Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
Steven
S
Johnson
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
Ariel J
AJ
Blocker
D000942:Antigens, Bacterial / Q000302:isolation & purification / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D015151:Immunoblotting; D008074:Lipoproteins / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry; D016253:Microscopy, Immunoelectron; D011485:Protein Binding; D021381:Protein Transport; D020539:Sequence Analysis, Protein; D012763:Shigella flexneri / Q000737:chemistry* / Q000378:metabolism; D037521:Virulence Factors / Q000302:isolation & purification* / Q000378:metabolism
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; C095770:IpaD protein, Shigella flexneri; D008074:Lipoproteins; D037521:Virulence Factors; C078384:mxiM protein, Shigella flexneri