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by adding the extracellular part of the Mid2 protein (
Fig. 15.7a
)
. In addition, a
His-tag was inserted to allow for speciic detection with AFM tips terminated
with NTA groups. With this strategy, single His-tagged elongated sensors
were detected on
. Adhesion force maps revealed
the localization of individual proteins, therefore conirming they were long
enough to reach the cell surface. By contrast, His-tagged Wsc1 that were not
elongated could not be detected, except in bud scars.
Notably, stretching single sensors revealed they behave like nanosprings,
capable to resist high mechanical force without undergoing secondary
structure unfolding (
Fig. 15.7b
)
. The sensor spring constant was estimated
to be ~5 pN nm
Saccharomyces cerevisiae
, which is very close to the behaviour of ankyrin repeats.
Lowering the salt concentration or increasing temperature resulted in a
substantial reduction of the sensor spring constant, indicating that Wsc1
is sensitive to cell surface stress. Both a genomic pmt4 deletion and the
insertion of a stretch of glycines in Wsc1 resulted in severe alterations in
protein spring properties, supporting the important role of glycosylation
at the extracellular serine/threonine-rich region. These indings have
pharmaceutical implications since drugs used in the treatment of fungal
infections are often directed against the protective fungal cell wall.
In the future, SMFS may help understanding how cell wall integrity is
maintained or altered upon interaction of the microbes with drugs. More
broadly, the combined method of genetic design and single-molecule
measurements used in this study has great potential for investigating how
proteins respond to forces in living cells and how mechanosensing events
proceed
1
in vivo
.
15.4 CONCLUSION
Our current view of microbial cell envelopes owes much to the development
of electron microscopy techniques. Yet, these methods cannot probe living
cells in buffer solution. The data surveyed here demonstrate the power of
AFM for imaging and force probing live cells down to molecular resolution.
These single-molecule assays complement traditional proteomic and
molecular biology approaches for the functional analysis of membrane and
cell wall proteins and may help in the search for novel anti-microbial drugs. In
particular, we anticipate that SMFS will ind valuable applications in clinical
microbiology and pathogenesis to investigate the interactions between
microbial pathogens and host cells and to localize cell surface receptors,
which may eventually help developing new therapeutic approaches.
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