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protected disaccharide alkyl conjugate. Deprotection of acetyl
groups with sodium methoxide followed by neutralization (to pH~
6.5) with Amberlite H+ resins (Zemplen deacetylation) yielded 5.
Synthesis of other maltose derivatives was conducted similarly (see
the Supporting Information).
Whether or not this is the case, we believe that maltose deriva-
tives could be promiscuous (binding to multiple bacterial pro-
teins).
We explored the potential of maltose derivatives to activate
P. aeruginosa quorum sensing circuits (las and/or rhl systems)
by using a previously established gene reporter strains (PAO1/
plasI-LVAgfp and PAO1/prhlI-LVAgfp) for identifying small mole-
cule quorum sensing inhibitor.[8] These reporter strains produce
natural AHL signals, and binding of these signal molecules to
the Lux-type receptor proteins (LasR and RhlR) activated the
expression of plasmid-encoded GFP. Our results indicate that
maltose derivatives did not compete with the natural signaling
molecules to cause a decrease in fluorescent signal (see the
Supporting Information). Furthermore, the ability of the mal-
tose derivatives to activate quorum sensing in P. aeruginosa in
the absence of natural AHLs was studied with double knock-
out strains PAO-JP2 (plasI-LVAgfp) and PAO-JP2 (prhlI-LVAgfP),
which do not produce AHLs.[9] The maltose derivatives did not
show any significant increase in the fluorescent signals (see
the Supporting Information), indicating that they did not ago-
nize the quorum sensing receptors.
Bacterial strains and growth media. Wild type P. aeruginosa PAO1
and PAO1-EGFP strains were obtained from Dr. Guirong Wang (Up-
state Medical University, Syracuse). The non-swarming P. aeruginosa
mutant, rhlA (PW6886, rhlA-E08::ISphoA/hah) was obtained from
the PA two-allele library (PAO1 transposon mutant library, Manoil
Lab, University of Washington Genome Sciences).[60] Strains PAO-
JP2 (plasI-LVAgfp) and PAO-JP2 (prhlI-LVAgfp) were obtained from
Dr. Helen E. Blackwell (University of Wisconsin-Madison). Plasmids
plasI-LVAgfp and prhlI-LVAgfp were obtained from Dr. Hiroaki Suga
(University of Tokyo). All bacterial strains were grown in lysogeny
broth (LB: tryptone (10 gLꢀ1), yeast extract (5 gLꢀ1), and NaCl
(10 gLꢀ1)) at 378C. For biofilm inhibition and dispersion assays
95% M9+ medium with 5% LB was used unless otherwise stated.
M9+ medium contained NH4Cl (18.7 mm), KH2PO4 (21.7 mm),
Na2HPO4 (47.7 mm), NaCl (8.6 mm), CaCl2 (0.1 mm), MgSO4 (1 mm),
anhydrous a-d(+)-glucose (0.2%), l-Arg (0.4%), citric acid mono-
hydrate (0.2%), casamino acids (0.5%), sodium succinate dibasic
hexahydrate (0.2%), and l-glutamic acid monopotassium salt
monohydrate (0.2%).[11]
Stock solutions of generic surfactants and maltose derivatives.
Stock solution of all the agents (11.5 mm) were prepared in auto-
claved water, sterilized by filtering through a 0.2 mm syringe filter,
and stored at ꢀ208C in sealed vials. Appropriate amounts of sterile
water were added to controls in all assays to eliminate solvent
effect.
Conclusions
This class of maltose derivative, with a wide range of aliphatic
chain structures, exhibited three versatile biological functions:
activation of swarming motility, inhibition of bacterial adhe-
sion, and inhibition of biofilm formation. Natural rhamnolipids
activated the swarming motility of a nonswarming mutant of
P. aeruginosa, rhlA; this series of maltose derivatives represent
the first class of synthetic molecules to activate the swarming
motility of this nonswarming mutant. Because the rhlA mutant
does not produce natural rhamnolipids, our results suggest
that the active maltose derivatives function as analogues of
rhamnolipids. As the bioactivities were highly sensitive to the
structural details of the agents, and because of the cross-inhib-
ition activities between the agents, this suggests that one or
more protein receptors exist for maltose derivatives as well as
rhamnolipids. As biofilm formation and swarming motilities are
common for a wide range of microbes, and as other bacteria
also produce rhamnolipids-like molecules,[31] these and other
disaccharide derivatives have potential anti-biofilm activity for
other microbial species. Because multiple biological activities
(adhesion, biofilm formation, and swarming) are affected by
a common set of molecular structures without killing the bac-
teria, this class of molecules might form the basis of an effec-
tive approach to control P. aeruginosa biofilm-related disease.
Swarming assay. Swarm agar plates were made with M8 medium
(Na2HPO4 (50 mm), KH2PO4 (25 mm), NaCl (4 mm)), supplemented
with glucose (0.2%), casamino acid (0.5%), MgSO4 (1 mm), and sol-
idified with Bacto agar (0.5%),[33] and inoculated with bacterial cul-
ture (3 mL, OD600 =0.4–0.6). Swarm agar plates were incubated at
378C for 12 h and then incubated for an additional 12 h at room
temperature. For each set of experiment all the swarm plates were
poured from same batch of agar and allowed to dry for 1 h before
inoculation. Each swarming experiment waas repeated at least
three times.
Effect of maltose derivatives on the growth of P. aeruginosa. Op-
tical density was measured with an ELx800 TM absorbance micro-
plate reader (BioTek Instruments, Inc., Winooski, VT) with Gen5TM
data analysis software. OD600 values were taken in sterile conditions
at 0, 2, 4, 6, 8, 10, 12, and 24 h after inoculation in 96-well polystyr-
ene plates, with or without agents in LB broth.
Crystal violet biofilm inhibition assay. The inhibitory effect of
maltose hydrocarbons on P. aeruginosa biofilm formation was de-
termined by crystal violet-dye-based biofilm inhibition assays.
Overnight culture of wild-type P. aeruginosa (PAO1) was subcul-
tured (initial OD600 =0.01) in M9+/LB (95:5) or LB. Aliquots (200 mL,
OD600 =0.1) of the subculture were placed in wells of a 96-well
polystyrene microtiter plate. Test compounds (concentrations as in-
dicated) were then added to the wells, and the plates were wrap-
ped in Press’n’Seal (GLAD, Oakland, CA) followed by incubation.
After (24 h, 378C), the medium was discarded, and the plates were
washed with water and dried (1 h, 378C). The plates were stained
with aqueous crystal violet (CV; 200 mL, 0.1%), followed by incuba-
tion (RT, 20 min). The CV stain was then removed, and the plates
were washed with water. Remaining biofilm-adhered stain was
resolubilized in acetic acid (200 mL, 30%). After the stain was dis-
Experimental Section
Organic synthesis of maltose derivatives. The maltose derivatives
were synthesized by a general organic synthetic route (Scheme 2).
As an example, we describe here the synthesis of benzyl dodecyl
b-maltoside, BDbM (5). Condensation of benzyl bromide with 1,12-
dodecanediol in presence of NaH yielded dodecanoyl benzyl ether.
Glycosidation of dodecanoyl benzyl alcohol with an acetobromo
maltose in the presence of an acid catalyst (FeCl3) gave the acetyl
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2014, 15, 1514 – 1523 1521