Transcript Effectiveness of 2 Chemical Inhibitors of a

Analysis of the Effectiveness of Three Chemical
Inhibitors of Alpha-Toxin in the Treatment of
S. aureus Experimental Keratitis
Armando R. Caballero, PhD1, Clare McCormick, PhD1, Vladimir Karginov, PhD2,
Richard O’Callaghan, PhD1.
1 The
authors have no financial interest in the subject matter of this poster
2 The
author has a financial interest in the subject matter of this poster
The authors wish to acknowledge the contribution of Aihua Tang and Anastasia Weeks to this research.
Purpose
S. aureus is a major cause of ocular infections worldwide. These include blepharitis,
keratitis, conjunctivitis and endophthalmitis (1-3). Keratitis and endophthalmitis can
result in the loss of visual acuity and blindness.
S. aureus secretes a variety of toxins which play an important role in its virulence.
In ocular settings, the most important virulence factor is alpha-toxin (4-6). Rabbit
and murine models of keratitis have shown that infection with an alpha-toxin mutant
strain produces significantly less ocular pathology than the parental strain (5).
Complementation of the alpha-toxin gene in the rescue strain restores full virulence.
Alpha-toxin is a 33 kDa protein that binds to caveolin receptors on lipid rafts,
forming a ring of seven alpha-toxin molecules that penetrate the host cell membrane
creating a pore that causes cell lysis (7). Alpha-toxin also up-regulates cytokines,
impairs host defenses by interfering with calcium flow, and can cause apoptosis (8-10).
S. aureus ocular infections can be successfully treated with antibiotics, however,
damage to the eye can continue for a time due to already secreted toxins (11).
There are no known inhibitors of alpha-toxin ready for clinical use, however, a methyl-cyclodextrin complexed with cholesterol can inhibit the toxin (12-14).
The purpose of this research is to determine the relative effectiveness of a
chemically modified form of methyl -cyclodextrin, methyl β-cyclodextrin cholesterol,
and methyl β-cyclodextrin alone in reducing the pathology associated with S. aureus
keratitis in a rabbit model of infection.
Methods
Bacteria: S. aureus strain 60171, a clinical ocular isolate was grown in TSB media
at 37°C overnight.
Animals: Specific pathogen free New Zealand white rabbits (from Harlan, Inc.; Indianapolis, IN) were maintained according to institutional guidelines and tenets of the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research. Rabbits were
anesthetized by subcutaneous injection of a 1:5 mixture of xylazine (100 mg/ml;
Rompum; Miles Laboratories, Shawnee, KS) and ketamine HCl (100 mg/ml; Ketaset;
Fort Dodge Animal Health, Fort Dodge, IA). Prior to intrastromal injection, proparacaine
HCl (0.5%; Bausch and Lomb, Tampa, FL) was topically applied to each eye. Rabbits
were euthanized by an intravenous overdose of pentobarbital (Sigma) into the ear vein.
Experimental Keratitis: Rabbit eyes (n ≥ 8 eyes per group) were injected with S. aureus
strain 60171 (100 CFU, 10 µl of TSB) into the corneal stroma. At 7 hours PI, a single
topical drop (45 l) of PBS, 1% methyl -cyclodextrin (CD), 1% methyl -cyclodextrincholesterol complex (CD-cholesterol), or 100 M modified methyl -cyclodextrin (M-CD)
was applied every 15 minutes from 7 to 8 hours PI and then every 30 minutes until 13
hours PI (total of 15 drops per group). At 13 hours PI, all eyes underwent slit lamp
examination (SLE) by two masked observers to quantify pathological changes.
Methods (cont’d)
SLE: Seven parameters were graded on a scale of 0 (normal) to a maximum of 4
(severe) using a Topcon SL-7E biomicroscope (Koaku Kikai K.K., Tokyo, Japan):
injection (redness), chemosis (swelling), iritis, hypopyon, corneal infiltrate, fibrin in the
anterior chamber, and corneal edema. The sum of these grades for each eye, after
averaging, determined the SLE score ± SEM, which could range from 0 (normal eye)
to a theoretical maximum of 28.
Colony forming unit determination: Corneas of rabbits infected with S. aureus
strain 60171 were harvested at 14 hours PI and homogenized in 3 ml sterile PBS.
The homogenates, as well as subsequent serial dilutions (1:10), were plated in
triplicate on TSA plates. Following incubation at 37°C for 24 hours, CFU per cornea
was determined and expressed as log CFU ± SEM.
Statistics: Statistical analyses were performed using statistical analysis software
(SAS, Cary, NC) or Microsoft Excel (Seattle,WA). For SLE results, statistical analyses
of inter-group differences were performed using non-parametric one-way analysis of
variance. For CFU determinations, analysis of variance and Student’s t tests between
least-squared means from each group were performed. P ≤ 0.05 was considered
significant.
Results: Virulence of alpha-toxin mutant and rescue strain in the
the rabbit eye
SLE SCORE
18
16
12
8
4
Parent
strain
Alpha-toxin
mutant
Rescue
strain
S. aureus strain 8325-4 was the alpha-toxin producing parental strain. An isogenic
mutant deficient in alpha-toxin was designated DU1090. A plasmid expressing
alpha-toxin (pDU1212) was introduced into DU1090 creating a rescue strain.
Infection with the alpha-toxin mutant strain resulted in significantly less ocular
pathology than the parental strain at 25 hours PI. Full virulence was restored in the
rescue strain by the plasmid expressing alpha-toxin (5).
Inhibitors of alpha-toxin
Methyl -cyclodextrin
A cyclic oligosaccharide
with a seven sugar ring
structure. It was made as
a 10% stock solution and
diluted to 1% in
PBS for treatment.
Cholesterol
A steroid metabolite.
Water soluble cholesterol
in methyl -cyclodextrin
was made as a 10%
solution and diluted to
1% in PBS for treatment.
Modified β-cyclodextrin
A methyl -cyclodextrin
molecule with side groups
at positions 6. It was
prepared in PBS as a
100 m solution for
treatment.
Inhibition of alpha-toxin hemolysis by modified β-cyclodextrin in vitro
1
2
3
4
5
6
7
8
9
10
11
12
A
B
C
Well A1 contains rabbit erythrocytes in PBS as a control showing settling of the
erythrocytes without any lysis. Wells A2 to B12 contain two-fold dilutions of alphatoxin. The alpha-toxin has a hemolytic titer of 1024 as evidenced by the lysis of the
erythrocytes in wells A2 -A11.
In Row C, rabbit erythrocytes were mixed with dilutions of modified methyl βcyclodextrin before the addition of alpha-toxin. The assay in row C shows a toxin
inhibition titer of greater than 4,096.
The inhibition of alpha-toxin hemolysis by cyclodextrin-cholesterol complex in vitro
has been previously demonstrated by McCormick et al. (12).
Effect of inhibitors of alpha-toxin on rabbit eyes infected with S. aureus
SLE SCORE
12
10
8
6
4
2
PBS
CD
CD
cholesterol
M-CD
Treatment of infected eyes with cyclodextrin (CD) did not significantly reduce ocular
pathology when compared to PBS treated eyes (p= 0.16449). Treatment with cyclodextrin-cholesterol complex (CD-cholesterol) significantly decreased ocular pathology
compared to PBS (p= 0.0005) and CD treated eyes (p= 0.005). Treatment with
modified -cyclodextrin (M-CD) reduced ocular pathology when compared to PBS
(p< 0.00005), CD (p< 0.00005), and CD-cholesterol treated eyes (p= 0.003).
Ocular pathology
PBS
Severe iritis,
injection, chemosis,
infiltrate and large
erosion.
Cyclodextrin
CD
cholesterol
Modified cyclodextrin
Severe iritis,
injection, chemosis,
infiltrate and large
erosion.
Moderate iritis,
injection and
chemosis. Small
erosion.
Moderate iritis,
injection and
chemosis. Trace
signs of erosion.
Bacterial load of S. aureus infected corneas treated with inhibitors of
alpha-toxin
8
LOG CFU
6
4
2
PBS
CD
CD
M-CD
cholesterol
There was no significant difference in the bacterial load per cornea among the
different treatment groups (p ≥ 0.347). This demonstrates that the decrease in
pathology was due exclusively to inhibition of alpha-toxin.
Conclusion
Methyl β-cyclodextrin cholesterol complex and modified methyl -cyclodextrin are
effective inhibitors of alpha-toxin in vivo. The CD-cholesterol molecule has previously
been shown to protect the cornea during experimental S. aureus keratitis (12).
The modified CD inhibitor has also been shown to have beneficial effects in treating
Staphylococcus pneumonia (15). The mechanism of inhibition has been ascribed
to the ability of the inhibitors to bind to the pore formed by the toxin which prevents
cell lysis (15).
The fact that the bacterial load was statistically the same in these corneas when
compared to the PBS treated controls demonstrates true interference with alphatoxin activity. Results from hemolysis assays in vitro using rabbit red blood cells,
purified alpha-toxin, and the inhibitors parallel the results seen in vivo.
Given the crucial role that alpha-toxin plays in the ocular pathology of S. aureus
keratitis, treatment with an inhibitor of alpha-toxin in combination with antibiotics
could prove to be an effective means of limiting the damage of the already secreted
toxin while the antibiotic kills the infecting bacteria.
References
1. Liesegang TJ. The Cornea. Boston, MA: Butterworth-Heineman; 1998:159-219.
2. Kattan HM, Flynn HW Jr, Pflugfelder SC, Robertson C, Forster RK. Nosocomial endophthalmitis survey. Current incidence
of infection after intraocular surgery. Ophthalmology. 1991;98(2):227-238.
3. McCulley JP, Shine WE. Changing concepts in the diagnosis and mangement of blepharitis. Cornea. 2000;19(5):650-658.
4. Callegan MC, Engel LS, Hill JM, O’Callaghan RJ. Corneal virulence of Staphylococcus aureus: roles of alpha-toxin and
protein A in pathogenesis. Infect Immun. 1994;62(6):2478-2482.
5. O’Callaghan RJ, Callegan MC, Moreau JM, et al. Specific roles of alpha-toxin during Staphylococcus aureus corneal infection.
Infect Immun. 1997;65(5):1571-1578.
6. Moreau JM, Sloop GD, Engel LS, Hill JM, O’Callaghan RJ. Histopathological studies of staphylococcal alpha-toxin: effects on
rabbit corneas. Curr Eye Res. 1997;16(12):1221-1228.
7. Valeva A, Weisser A, Walker B, et al. Molecular architecture of a toxin pore: a 15 residue sequence lines the transmembrane
channel of staphylococcal -toxin. EMBO J. 1996;15(8):1857-64.
8. Liang X, Ji Y. Involvement of alpha5beta1-integrin and TNF-alpha in Staphylococcus aureus alpha-toxin-induced death of
epithelial cells. Cell Microbiol. 2007;9(7):1809-21.
9. Hasliner B, Strangfeld K, Peters G, Schulze-Osthoff K, Sinha B. Staphylococcus aureus alpha-toxin induces apoptosis in
peripheral blood mononuclear cells: role of endogenous tumor necrosis factor-alpha and the mitochondrial death pathway.
Cell Microbiol. 2003;5(10):729-41.
10. Haslinger-Löffler B, Kahl BC, Grundmeier M, et al. Multiple virulence factors are required for Staphylococcus aureus-induced
apoptosis in endothelial cells. Cell Microbiol. 2005;7(8):1087-97.
11. O’Callaghan RJ. Role of exoproteins in bacterial keratitis: the Fourth Annual Thygeson Lecture, presented at the Ocular
Microbiology and Immunology Group Meeting, November 7, 1998. Cornea. 1999;18(5):532-7.
12. McCormick C, Caballero A, Balzli C, Tang A, O’Callaghan R. Chemical inhibition of alpha-toxin, a key corneal virulence factor
of Staphylococcus aureus. IOVS. 2009;50:2848-2854.
13. Gu LQ, Bayley H. Interaction of the noncovalent molecular adapter, beta-cyclodextrin, with the staphylococcal alpha-hemolysin
pore. Biophys J. 2000;79(4):1967-75.
14. Karginov VA, Nestorovich EM, Schmidtmann F, et al. Inhibition of S. aureus alpha-hemolysin and B. anthracis lethal toxin by
beta-cyclodextrin derivatives. Bioorg Med Chem. 2007;15(16):5424-31.
15. Ragle B, Karginov V, Wardenburg J. Prevention and treatment of Staphylococcus aureus pneumonia with a -cyclodextrin
derivative. Antimicrob Agents Chemother. 2010;54(1): 298-304.