3
124
TSUBERY ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
sion of 7 ϫ 106 cells/ml of Hanks balanced salt solution was placed in the upper
TABLE 2. Effects of peptides and peptide conjugates on
sensitivities of gram-negative bacteria to novobiocin
–
5
compartment and a 50-g/ml (ϳ3.5 ϫ 10 M) solution of the test peptide was
placed in the lower compartment of a 5-m membrane filter chamber. After
incubation for 3 h at 37°C, the number of PMNs per microscopic field that
crossed the 5-m membrane filter from the chamber’s upper to lower compart-
ment was enumerated under oil immersion (magnification, ϫ1,000) for 10 fields.
Opsonophagocytosis assays. The binding of the bacteria to phagocytic cells
and their subsequent destruction were determined by a modified version of a
method described previously (2). The indicated concentrations of the test pep-
tides in 0.05 ml RPMI 1640 or the buffer alone (control) were added to an
a
and erythromycin
MIC (g/ml)
Peptide
Novobiocin
KP
Erythromycin
EC
PA
EC
KP
None
Ͼ125
Ͼ250
Ͼ1,000 Ͼ250
Ͼ250
PMBN
1
4
ND
125
125
ND
250
250
ND
8
16
1
ND
8
0.5
4
8
8
16
6
equivalent volume (0.5 ml) of a mixture of phagocytic (2 ϫ 10 /ml) and bacterial
PMBNs
2
2
0.8
0.8
32
8
(
5 ϫ 10 /ml) cell suspensions in buffer. The mixture was rotated end over end at
[
[
fMLF]PMBN
nfMLF]PMBN
PMEN
fMLF]PMEN
0.12
0.5
2
2
2
5 rpm at 4°C for 30 min and washed three times with the buffer by centrifugation
ND
8
at 300 ϫ g for 5 min to remove the unbound bacteria. The phagocytic cell pellet
was resuspended in buffer, its temperature was raised to 37°C, and 0.1-ml sam-
ples were withdrawn after 30 min to determine the number of bacteria associated
with the phagocytic cells and after 3 h to determine the destruction of the
bacteria by the phagocytes. To estimate the number of bound bacteria, the
samples were distributed onto glass coverslips (22 by 22 mm) and placed in small
petri dishes (35 mm in diameter) at RT for 15 min to allow the phagocytic cells
to adhere onto the glass surfaces. The coverslips were then washed once in
buffer, removed from the petri dishes with forceps, rinsed briefly in 0.5% NaCl,
mounted on slides, dried vertically, fixed with methanol, and stained with He-
macolor (E. Merck). Two hundred cells were counted to determine the average
number of bacteria per phagocytic cell. To estimate the intracellular destruction
of the associated bacteria, the samples were disrupted in 1 ml distilled water,
diluted serially 10-fold, plated on MacConkey agar plates, and incubated over-
night at 37°C. The number of CFU was then determined.
[
0.5
2
4
0.25
1
1
PMBO
PMBOs
4
4
4
62
62
8
[
[
nfMLF]PMBO
1
ND
Ͼ250
ND
Ͼ250
2
,4,7,8
b
Lys
]PMBN Ͼ125
Ͼ250
ND
a
The MICs of novobiocin and erythromycin were determined in the absence
none) or presence of 30 g/ml (ϳ25 ϫ 10Ϫ6 M) of the indicated peptide for E.
(
coli and K. pneumoniae and 16 g/ml for P. aeruginosa, as described in the text.
Values are the means of at least six determinations, and in no case did the values
exceed 1 dilution. Abbreviations: EC, E. coli; KP, K. pneumoniae; PA, P. aerugi-
nosa; ND, not determined.
A control peptide consisting of a PMBN analog with lysine substitution has
a reduced affinity for LPS (28).
b
Acute toxicity. Solutions of test peptides (0.2 ml in sterile saline, administered
with a needle 0.5 by 16 mm) were intravenously (i.v.) injected into the tail veins
of male CD1 mice (age, 4 to 6 weeks; weight, 24 to 26 g; n ϭ 5 mice per group).
Survival was monitored after 1 day, and the amount of test peptide per kg of
Determination of MIC. Clinical isolates of Escherichia coli (EC1), Klebsiella
pneumoniae (K2), and Pseudomonas aeruginosa (33347) were obtained as de-
scribed elsewhere (19). The gram-negative bacteria were grown on nutrient agar
plates (Difco Laboratories, Detroit, Mich.) and kept at 4°C. Lyophilized aliquots
of the test peptides (2 mg; determined by weight and ascertained by amino acid
composition analysis) were dissolved in sterile, double-distilled water and filtered
by using a 0.2-m-pore-size Acrodisc. The number of CFU in an overnight
mouse body weight that constituted a lethal dose for 50% of the animals (LD50
)
was calculated.
Mouse protection assays. The ability of the PMBN, the PMBN-based peptide
conjugates, and PMB to protect mice from a lethal dose of erythromycin-resis-
tant K. pneumoniae was determined as described previously (19). Briefly, 0.5 ml
5
of bacterial suspension (2 ϫ 10 CFU/ml) was intraperitoneally (i.p.) injected
culture of bacteria in Isotonic Sensitest Broth (ISB; Oxoid) was adjusted to 1 ϫ
5
into male ICR mice (weight, 18 to 20 g; n ϭ 6 per group). Four hours after
bacterial inoculation (i.e., on day 1 at 4 h), each of three groups of mice was i.v.
injected with erythromycin, PMB, or PMBN alone. Also at 4 h, other experi-
mental groups of mice received an i.v. peptide-antibiotic mixture containing
PMBN, [fMLF]PMBN, or [nfMLF]PMBN (4 mg/kg of mouse body weight)
together with erythromycin (10 mg/kg of mouse body weight) in 0.5 ml of
phosphate-buffered saline. On the following day (day 2), each group received the
same treatment given on day 1, except that the doses were split in half, with the
second dose being delivered 4 h after the first one. Thus, the experimental mice
received two i.v. injections of test peptide (2 mg/kg) and/or erythromycin (5
mg/kg mice), as appropriate, 4 h apart on day 2. On day 3 the mice were injected
in the same manner once more. Survival was monitored daily for 7 days.
Blood clearance in mice. The ability of PMEN, [fMLF]PMEN, and PME to
1
0
CFU/ml and inoculated onto microtiter plate wells, each of which contained
1
00 l of a serial twofold dilution (1,000 to 0.5 g/ml) of the tested antibiotics or
peptides in ISB. The MIC was defined as the lowest peptide concentration at
which there was no visible bacterial growth after incubation for 20 h at 37°C. The
results (Table 2) are reported for three to five separate tests, which varied by no
more than 1 dilution.
Permeabilizing activity. The ability of the test peptides to permeabilize the
membranes of the gram-negative bacteria was determined as described else-
where (19). Briefly, a bacterial suspension (10 l; 1 ϫ 105 CFU) was inoculated
onto microtiter plate wells containing 100 l of a serial twofold dilution (1,000 to
0
.5 g/ml) of novobiocin or erythromycin (Sigma Chemical Co.) in ISB. To each
well, 10 l of test peptide was added, to achieve a final test peptide concentration
of 50 g/ml. The extent to which the MIC of novobiocin or erythromycin de-
creased between wells, in the presence or the absence of the test peptides, was
calculated and was designated the peptide’s permeabilizing activity.
enhance the clearance of bacteria from blood was determined by i.v. injection of
6
0
.5 ml saline containing 10 CFU K. pneumoniae into the tail veins of groups of
four mice. At 2.5 min, a blood sample was taken from the eye. The mice were
then immediately injected with 0.5 ml saline alone (group a) or with saline
containing 2 mg/kg of mouse body weight (ϳ40 g/mouse) of PMEN (group b),
a mixture of PMEN and fMLF (group c), [fMLF]PMEN (group d), or PME
LPS binding assay. The binding of the test peptides to bacterial LPS and the
latter’s affinity for the peptides were determined as described previously (27).
Briefly, the fluorescence of dansyl-PMBN bound to E. coli LPS was measured
with an MC200 monochromator (SLM AMINCO; SLM Instruments, Inc.) set at
an excitation wavelength of 340 nm and at an emission wavelength of 485 nm.
Binding affinity was evaluated by determining the concentration of the test
peptide required to displace dansyl-labeled PMBN from LPS. To a quartz cu-
(group e). At various time intervals thereafter, a 0.02-ml blood sample was
withdrawn from the eye with a disposable pipette, diluted 10-fold with distilled
water, and plated on nutrient agar to determine the number of CFU/ml blood.
Ϫ7
vette containing LPS solution (2 ml; 3 g/ml; ϳ2 ϫ 10 M) in HEPES buffer (5
mM; pH 7.2), dansyl-PMBN (0.55 M) was added and was allowed to equilibrate
at RT for 10 to 15 min. Subsequently, small portions (5 to 10 l) of test peptide
solution (1 ϫ 10 to 1 ϫ 10 M) were added. The inhibition of fluorescence
was measured 5 min after each addition. The percent inhibition was plotted as a
function of the peptide concentration, and 50% inhibitory concentrations were
calculated from the maximal specific displacement.
RESULTS
Ϫ5
Ϫ3
Membrane permeabilization of gram-negative bacteria by
PMBN conjugates. Previous studies have shown that PMBN
and PMBO increase OM permeability for gram-negative bac-
teria by binding to the bacterial surface (15, 31, 32). The test
peptides lacked bactericidal activity at concentrations Ն250
g/ml (data not shown). Significantly, the MICs of the formyl
peptide conjugate ([fMFL]PMBN) were 250 to 500, 500, and
250 to 125 g/ml against E. coli, K. pneumonia, and P. aerugi-
Preparation of phagocytic cells. Two types of phagocytic cells were employed.
6
Human PMN suspensions (10 cells/ml) were prepared as described previously
6
(
14, 20), and a suspension of mouse peritoneal macrophages (10 cells/ml) was
prepared as described elsewhere (2, 21).
Chemotactic activity. The chemotactic activity of the test peptides toward
PMNs was assayed as described previously (20). Briefly, a human PMN suspen-
Tsubery, Haim
