There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.
Abstract
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.