Visualizing bacterial surfaces in real time
Article first published online: 18 FEB 2009
© 2009 The Authors. Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd
Special Issue: Bioremediation. With guest editors Jan Roelof van der Meer, Thomas Wood, Hideaki Nojiri, Pieter van Dillewijn
Volume 2, Issue 2, pages 146–147, March 2009
How to Cite
Covacci, A. and Rappuoli, R. (2009), Visualizing bacterial surfaces in real time. Microbial Biotechnology, 2: 146–147. doi: 10.1111/j.1751-7915.2009.00090_14.x
- Issue published online: 18 FEB 2009
- Article first published online: 18 FEB 2009
The study of the network of proteins, protein complexes, sugars and surfaces organelles has been frustrated by the complexity of bacterial surfaces and the necessity to rely on molecular coordinates. The logical consequence to develop crystallographic methods for large cellular components and the alternative use of cryo-electron microscopy to solve organelles structure (flagellum, Type IV pili) has been fundamental but inherently slow. Development of new vaccines may also depend on the identification of complexes located at the surface and exposed protective molecules. Prediction of both is part of routine genome analysis using dedicated algorithms. Antibody staining and FACS analysis is a potent tool to visualize exposed molecules while it is heavily dependent from antibody specificity, affinity and outer layer penetration effect (detection of hidden structures by antibody penetration during preparation of the sample).
We suggest it is now possible to merge high-resolution fluorescence and scanning confocal microscopy with sortase tagging (Popp et al., 2007) to generate native molecules bearing a chromophore. This will potentially allow image rendering of the bacterial surface to visualize the dynamics of protein topology during growth and infection in real time. Science and Nature have both expressed their wonder in 2008 about recent advances in light microscopy (Chi, 2008). In addition, white light sources are moving from lasers to inexpensive mass production of LED (http://www.lens.unifi.it). White light is a necessary step to pulse a sample with a wide range of light wavelengths to simultaneously collect signals in the visible spectrum from excited dyes. The resulting live image can integrate all labelled proteins in a single picture and with a different colour tag providing realistic 3D coordinates. The crucial step in closing the loop is in vivo tagging of a target protein. This in theory can provide a repertoire of one strain-one tagged protein with the final goal to colour-code all the proteins of a bacterial species.
Sortases are bacterial enzymes that predominantly catalyse the attachment of surface proteins to the bacterial cell wall (Telford et al., 2006; Popp et al., 2007). Other sortases polymerize pilin subunits for the construction of the covalently attached pili of the Gram-positive bacteria (Telford et al., 2006). The sortase recognition sequence of Staphylococcus aureus sortase A, LPXTG, when engrafted near the C-terminus of proteins without natural sortase specificity, should be part of a sortase-catalysed transpeptidation reaction using artificial glycine-based nucleophiles. The chemical modification of such substrates with fluorophores allows modifications of proteins in in vitro and in vivo conditions. This method can be efficiently scaled-up for high-throughput data capture. Once the expected wave of new microscopes will be available and tagging with fluorophores will be pervasive, this technology should be ready also for fast and unexpensive real-time expression analyses.