1 / 12

Analysis of the Effectiveness of Three Chemical Inhibitors of Alpha-Toxin in the Treatment of S. aureus

Analysis of the Effectiveness of Three Chemical Inhibitors of Alpha-Toxin in the Treatment of S. aureus Experimental Keratitis Armando R. Caballero , PhD 1 , Clare McCormick, PhD 1 , Vladimir Karginov, PhD 2 , Richard O’Callaghan, PhD 1 .

piera
Download Presentation

Analysis of the Effectiveness of Three Chemical Inhibitors of Alpha-Toxin in the Treatment of S. aureus

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 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.

  2. 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.

  3. 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.; Indiana- polis, 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 -cyclodextrin- cholesterolcomplex (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.

  4. 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, fibrinin 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.

  5. Results: Virulence of alpha-toxin mutant and rescue strain in the the rabbit eye 18 16 12 SLE SCORE 8 4 Alpha-toxin mutant Parent strain 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).

  6. Inhibitors of alpha-toxin Methyl -cyclodextrin Modified β-cyclodextrin Cholesterol A methyl -cyclodextrin molecule with side groups at positions 6. It was prepared in PBS as a 100 m solution for treatment. A steroid metabolite. Water soluble cholesterol in methyl -cyclodextrin was made as a 10% solution and diluted to 1% in PBS for treatment. 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.

  7. 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 alpha-toxin. 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 β-cyclodextrin 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).

  8. Effect of inhibitors of alpha-toxin on rabbit eyes infected with S. aureus 12 10 8 SLE SCORE 6 4 2 PBS CD CD M-CD cholesterol Treatment of infected eyes with cyclodextrin (CD) did not significantly reduce ocular pathology when compared to PBS treated eyes (p= 0.16449). Treatment with cyclo- dextrin-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).

  9. Ocular pathology Modified - cyclodextrin CD cholesterol PBS Cyclodextrin Severe iritis, injection, chemosis, infiltrate and large erosion. Severe iritis, injection, chemosis, infiltrate and large erosion. Moderate iritis, injection and chemosis. Small erosion. Moderate iritis, injection and chemosis. Trace signs of erosion.

  10. Bacterial load of S. aureus infected corneas treated with inhibitors of alpha-toxin 8 6 LOG CFU 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.

  11. 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 alpha- toxin 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.

  12. 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 Staphylococcusaureus-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.

More Related