Biological Activities of Lasso Peptides and Structure–Activity Relationships - Lasso Peptides: Bacterial Strategies to Make and Maintain Bioactive Entangled Scaffolds - page 57

MccJ25 has the most potent antibacterial activity among lasso peptides (Vincent
and Morero 2009 ). It is active against bacteria phylogenetically related to the
producing strain ( Enterobacteriaceae such as certain Escherichia, Salmonella
and Shigella species) and shows minimal inhibitory concentrations (MICs) in
the nanomolar range against Salmonella (Table 3.2 ). It is the lasso peptide that is
best characterized in terms of mechanism of action. Its antibacterial activity re-
lies on (1) uptake by the target bacteria, which involves the outer membrane iron-
siderophore receptor FhuA (Salomón and Farías 1993 ; Destoumieux-Garzón et al.
2005 ; Mathavan et al. 2014 ), the inner-membrane energy transduction complex
TonB-ExbB-ExbD and the inner-membrane protein SbmA (Salomón and Farías
1995 ; de Cristóbal et al. 2006 ), followed by (2) inhibition of the bacterial RNAP
(Delgado et al. 2001 ; Yuzenkova et al. 2002 ; Adelman et al. 2004 ; Mukhopadhyay
et al. 2004 ; see Sect. 3.1.2).
FhuA is a 79-kDa outer-membrane siderophore receptor, which transports Fe(III)
chelated to the hydroxamate siderophore ferrichrome in E. coli (Chakraborty et al.
2007 ). It is a monomeric β-barrel protein consisting of 22 antiparallel β-strands. Its
N-terminus folds inside the β-barrel from the periplasmic side, forming the cork
domain (residues 20-160), and a large extracellular ligand-binding pocket open
to the external medium (Locher et al. 1998 ). Following recognition, transport by
FhuA uses energy that is provided by the proton motive force and transduced by the
TonB/ExbB/ExbD complex (called the Ton system), located at the inner membrane
(Braun and Endriss 2007 ; Postle and Larsen 2007 ). Energy transduction from the
inner membrane to FhuA involves contacts established in the periplasm between
a TonB region called the TonB box and FhuA (Killmann et al. 2002 ; Carter et al.
2006 ). Besides its essential role in iron uptake, FhuA can be hijacked for uptake
by the siderophore-conjugated antibiotic albomycin (Braun 1999 ; Ferguson et al.
2000 ), a structural analogue of ferrichrome, but also by antibiotics and antimicro-
bial peptides with no structural similarity with ferrichrome, such as rifamycin, CGP
4832 (Pugsley et al. 1987 ; Ferguson et al. 2001 ) and colicin M (Killmann et al.
1995 ). It is also the receptor for phages T1, T5 and Φ80 (Killmann et al. 1995 ;
Bonhivers et al. 1998 ). The interaction between the viral receptor-binding protein
(rbp) and FhuA results ultimately in the phage DNA release in the host cytoplasm
(Flayhan et al. 2012 ). As for its conventional role in iron uptake, the hijacked activ-
ity of FhuA requires the Ton system, except for phage T5 (Braun et al. 2002a , b ).
Mutants of E. coli resistant to MccJ25 permitted to propose that FhuA and the
Ton system are involved in the uptake of the peptide in the target bacteria (Salomón
and Farías 1993 , 1995). The role of FhuA in MccJ25 uptake was confirmed and
further characterized in 2005 (Destoumieux-Garzón et al. 2005 ). MccJ25 binding
to FhuA was shown by size exclusion chromatography and isothermal titration calo-
rimetry ( K d 1.2 µM, 2:1 stoichiometry). MccJ25 inhibited phage infection by phage
T5 in E. coli, suggesting that MccJ25 and the viral rbp5 (Flayhan et al. 2012 ) com-
pete for FhuA binding. Binding to FhuA was altered and antibacterial activity was
significantly lowered for MccJ25 cleaved within the Val11-Pro16 region by thermo-
lysin (Rosengren et al. 2004 ; Destoumieux-Garzón et al. 2005 ), indicating that the
loop region of MccJ25 is required for recognition by FhuA. The structure of FhuA