Biological Activities of Lasso Peptides and Structure–Activity Relationships - Lasso Peptides: Bacterial Strategies to Make and Maintain Bioactive Entangled Scaffolds - page 62

This method is based on an orthogonal control of the expression of mcjA and mcjD,
permitting independent control of MccJ25 production and export/immunity. Site-
directed mutagenesis studies have been performed in our group to generate a series
of variants specifically designed with varying sizes of macrolactam ring, loop and
C-terminal tail below the ring, aiming at deciphering the residues that are critical
for both the lasso fold and the antibacterial activity (Ducasse et al. 2012b ). This
study was completed by a characterization of the topology of the variants generated,
which permitted to discriminate lasso and branched-cyclic peptides. The size of the
loop revealed critical for preserving the antibacterial activity, due to its role in the
interaction with FhuA (Ducasse et al. 2012b ). The C-terminal tail could be extended
while preserving antibacterial activity, but for the normal length peptide, the nature
of the C-terminal residue appeared essential for the antimicrobial activity: Asp or
Asn residues allow maintaining a weak activity, while Arg, Lys, Glu or Tyr residues
result in a total loss of activity. Finally, the His5 residue has revealed critical for
MccJ25 uptake, being involved in both FhuA binding (Mathavan et al. 2014 ) and
SbmA-mediated entry (de Cristóbal et al. 2006 ). Synthetic peptides derived from
the sequence of MccJ25, designed to form a compact conformation maintained by
disulfide bridges, showed a weak antibacterial activity against Salmonella strains
for one peptide, through inhibition of cell respiration (Soudy et al. 2012 ). This sug-
gests that inhibition of the membrane respiratory chain, which constitutes a second-
ary mechanism of MccJ25 activity against Salmonella and Escherichia that requires
higher concentration of MccJ25, does not necessitate the lasso topology.

3.2.2 Parameters Governing the Activity Spectrum

MccJ25 does not induce inhibition of yeast RNAP II and RNAP III, nor of RNAP
from the Gram-positive bacteria Bacillus subtilis and the thermophilic Gram-
negative Thermus aquaticus (Yuzenkova et al. 2002 ). This trend suggests selectivity
in the activity of MccJ25, in accordance with its narrow spectrum of antibacterial
activity. However, the main factor governing the activity spectrum of antibacte-
rial lasso peptides is most probably the uptake in target cell. Differences in FhuA
sequence within Gram-negative bacteria may account for the narrow spectrum of
activity of MccJ25. Indeed, Salmonella typhimurium, which is totally resistant to
MccJ25, becomes highly sensitive when expressing E. coli FhuA (Vincent et al.
2004 ), while a FhuA-defective E. coli expressing wild-type FhuA of Salmonella
typhimurium became resistant to MccJ25 (Killmann et al. 2001 ). In addition, the
combination of MccJ25 to a membrane-permeabilizing peptide (KFF) 3 K allowed
MccJ25 penetration in an FhuA and SbmA-independent manner, extending the
spectrum of activity towards pathogenic Salmonella strains such as Salmonella ty-
phimurium (Pomares et al. 2010 ). The fact that RNAP is a common intracellular
target for MccJ25 and capistruin, two lasso peptides that exhibit a different spec-
trum of activity (Kuznedelov et al. 2011 ), also supports this idea. All these elements
indicate that the narrow spectrum of activity of lasso peptides is due to specific