.
Angewandte
Communications
Table 2: MIC values and oxacillin resistance suppression activity of
compound 7d against strains from the Nebraska Transposon Mutant
Library.
isolates obtained from the ATCC (see the Supporting
Information) and observed reduction in MIC values of
4-fold to 512-fold.
Entry Strain Concentration Oxacillin MIC Oxacillin MIC with 7d
As the synthesized molecules are amphipathic, we first
investigated the effect of the compounds on cell-membrane
integrity. The ability of compound 7d to permeabilize the
bacterial cell membrane was quantified using the BacLight
assay.[16] After exposure of strain JE2 to compound 7d for one
hour, the ratio of intact/damaged cells was measured and
compared to control (treated with DMSO only) bacteria. At
4 ꢁ the MIC, 96% of cells were damaged, whereas at 1 ꢁ ,
0.4 ꢁ , and 0.25 ꢁ the MIC, only 33%, 21%, and 9% of cells
were damaged, respectively. An inactive compound (7e) was
found to be comparable, with 83%, 24%, 23%, and 16% of
cells damaged at 4 ꢁ , 1 ꢁ , 0.4 ꢁ , and 0.25 ꢁ the MIC,
respectively; this suggests that cell-membrane permeabiliza-
tion is not the mechanism by which compound 7d suppresses
resistance to oxacillin. Importantly for a potential antibiotic
adjuvant, 7d exhibited little effect on eukaryotic cell mem-
branes, as determined by measuring the hemolytic activity
against mechanically difibrinated sheep blood.[9] At its active
resistance suppression concentration (5 mm), less than 1%
lysis was observed compared to triton ꢁ positive control,
while only 5.6% lysis was observed at as high as 50 mm.
To further delineate the mechanism by which these 2-AIT
conjugates are able to lower the oxacillin MIC against MRSA,
we obtained a number of mutant strains belonging to the
Nebraska Transposon Mutant Library from the Network on
Antimicrobial Resistance in S. aureus (NARSA). These
mutants are all derived from JE2, thus allowing us to probe
non-essential pathways that may be involved in suppression of
oxacillin resistance. For this screen, we focused largely on
mutants of non-essential TCS. Bacterial TCS, consisting of
a membrane-bound histidine kinase and a response regulator,
regulate adaptation to environmental changes and have been
shown to play a role in resistance to certain antibiotics[17–19] as
well as being master regulators of biofilm formation.[20,21]
Furthermore, biotinylated analogues of related 2-AI anti-
biofilm compounds employed in pull down assays bind to
response regulators involved in biofilm formation (unpub-
lished work). Therefore, we posited that these 2-AIT deriv-
atives, which also have anti-biofilm activity, might also target
other response regulators involved in antibiotic resistance.
We first established the MICs of oxacillin and 7d against
each mutant strain (Table 2; see the Supporting Information
for gene descriptions). As expected, the MIC of 7d was fairly
consistent against all mutant strains (either 6.25 or 12.5 mm),
whereas a majority of the strains examined, including several
response regulator mutants (strains NE958, NE481, NE262,
and NE49), histidine kinase mutants (strains NE218, NE147,
NE618, NE873, NE820, NE116, and NE423), and a MecR1
regulatory protein mutant (strain NE839), did not exhibit
a greater than two-fold difference in oxacillin MIC compared
to the parent strain. However, three of the strains tested
exhibited considerably lower oxacillin MIC values: NE481
(an unidentified DNA-binding response regulator mutant),
NE554 (a vraR mutant), and NE823 (a vraS mutant)
exhibited oxacillin MICs that were reduced 16-fold, eight-
fold, and eight-fold, respectively. These results are in line with
of 7d [mm]
[mgmLÀ1
]
[mgmLÀ1 [a]
]
1
2
3
4
5
6
7
8
JE2
NE218 2.5
NE147 2.5
NE958
NE481
NE262
NE618
NE554 2.5
NE823 2.5
5
32
32
32
32
2
32
32
4
0.5
8
0.25
0.25
0.25
0.5
1
5
5
5
5
4
4
9
4
10
11
12
13
14
15
16
17
NE873
NE210
NE820
NE839
NE49 2.5
NE116 2.5
5
5
5
5
32
32
32
32
32
32
16
16
0.25
0.5
0.25
0.5
16
32
0.5
4
NE95
NE423 2.5
5
[a] Oxacillin MIC values recorded in the presence of 40% MIC of 7d.
previous studies which show that expression of VraSR
contributes to oxacillin resistance.[22] The ability of 7d to
lower the oxacillin MIC against the mutant strains was then
examined in an identical manner to that used for the parent
strain (at 40% of the MIC). Of the mutants that exhibited
altered oxacillin MIC values versus the parent, compound 7d
failed to lower the MIC of both the VraSR two-component
system mutant strains NE554 and NE823, which suggests that
the mode of action of the oxacillin resistance suppression
activity of compound 7d involves VraSR. Compound 7d also
failed to lower the oxacillin MIC by more than two-fold
against strains NE116 and NE49, which suggests that com-
pound 7d may have some interaction with the pathways
controlled by the disrupted genes of these two mutants.
NE116 is a putative histidine kinase mutant, whereas NE49 is
an AraC family response regulator mutant. AraC family
proteins are known to play a role in antibiotic resistance and
stress responses,[23] however, as these two mutant strains did
not exhibit oxacillin MICs that differed from the parent
strain, these pathways most likely do not relate to the
mechanism of oxacillin resistance suppression by compound
7d.
As compound 7d exhibited a lower MIC against a number
of mutant strains than the parent strain (and was therefore
screened for resistance suppression at a lower concentration,
2.5 mm, 1.55 mgmLÀ1), we wanted to ensure that the lack of
resistance suppression activity was not simply a result of lower
bactericidal activity of the compound. A time-kill curve was
therefore constructed for NE554 in the presence of 2.5 mm 7d
(40% MIC) and compared to the time-kill curve of JE2 in the
presence of 5 mm 7d (see the Supporting Information). The
bactericidal activity of compound 7d, was in fact slightly
higher against strain NE554 than JE2 at the concentrations
used in the resistance suppression assay, which suggests that
the lack of activity against NE554 is due to the absence of
VraR, rather than altered bactericidal activity.
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 11254 –11257