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Evaluation of the Role of Shiga and Shiga-Like Toxins in Mediating Direct Damage to Human Vascular Endothelial Cells

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Evaluation of the Role of Shiga and Shiga-Like Toxins in Mediating Direct Damage to Human Vascular Endothelial Cells University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Uniformed Services University of the Health Sciences U.S. Department of Defense 1991 Evaluation of the Role of Shiga and Shiga-like Toxins in Mediating Direct Damage to Human Vascular Endothelial Cells Vernon L. Tesh Uniformed Services University of the Health Sciences James E. Samuel Biocarb, Inc. Liyanage P. Perera Uniformed Services University of the Health Sciences John B. Sharefkin Uniformed Services University of the Health Sciences Alison D. O'Brien Uniformed Services University of the Health Sciences, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/usuhs Part of the Medicine and Health Sciences Commons Tesh, Vernon L.; Samuel, James E.; Perera, Liyanage P.; Sharefkin, John B.; and O'Brien, Alison D., "Evaluation of the Role of Shiga and Shiga-like Toxins in Mediating Direct Damage to Human Vascular Endothelial Cells" (1991). Uniformed Services University of the Health Sciences. 113. https://digitalcommons.unl.edu/usuhs/113 This Article is brought to you for free and open access by the U.S. Department of Defense at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Uniformed Services University of the Health Sciences by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 344 Evaluationof the Role of Shiga and Shiga-like Toxins in Mediating Direct Damage to Human Vascular Endothelial Cells Vernon L. Tesh, James E. Samuel,* Liyanage P. Perera, Departmentsof Microbiologyand Surgery, UniformedServices University John B. Sharefkin, and Alison D. O'Brien of the Health Sciences, Bethesda, Maryland Infectionwith Shiga toxin- andShiga-like toxin-producing strains of Shigelladysenteriae and Escherichiacolif respectively,can progressto the hemolytic-uremicsyndrome. It has been hy- pothesizedthat circulatingShiga toxin, Shiga-liketoxins, and endotoxins may contribute to the diseaseby directlydamaging glomerular endothelial cells. The effectsof these toxins on HeLa, Vero,and human vascular endothelial cells (EC)were examined. Confluent EC weresensitive to Shigatoxin butwere at least 106-fold less sensitiveto the toxinsthan were Vero cells. Shigatoxin was the predominantcytotoxic factor. Lipopolysaccharides were not cytotoxicand did not aug- mentShiga toxin-mediated toxicity. Lower doses of Shigatoxin causedcytotoxicity when coin- cubatedwith tumornecrosis factor. The relativeresistance of EC to Shigatoxin and Shiga-like toxins may be due to reducedtoxin binding,as low levels of globotriaosylceramide(Gb3), the toxin-specificreceptor, were found in EC membranes. Inflammatorybacterial colitis caused by infection with from the 28S rRNA component of the 60S eukaryoticribo- Shigella species continues to be a major cause of morbidity some complex [8, 9]. The B subunit mediatesbinding of the and mortality in underdeveloped countries, especially holotoxin to specificglycolipid receptorsin mammaliancell among young children [1]. Of particularinterest is Shigella membranes [10-13]. Although the precise role of Shiga dysenteriaetype 1, which is capable of causingepidemic out- toxin and the SLTsin the pathogenesisof bacillarydysentery breaksof dysentery.In addition, it has recently been shown and hemorrhagiccolitis, respectively, is not fully under- that some strainsof Escherichiacoli, particularlyE. coli sero- stood, there is now convincing evidence to suggest that the type 0157:H7, can cause hemorrhagiccolitis [2, 3]. Collec- toxins are importantvirulence factors in the developmentof tively, these E. coli strains are referredto as enterohemor- bloody, edematous vascular lesions of the colon [14]. The rhagicE. coli (EHEC). S. dysenteriaetype 1 and EHECshare toxins may also participatein the direct killing of colonic the characteristicof producinghigh levels of protein toxins epithelial cells and may provokefluid secretionand diarrhea called Shigatoxin or Shiga-liketoxins (SLTs or Verotoxins), in the host [7]. respectively.EHEC may produce two antigenicallydistinct Perhaps the most serious sequela of colitis caused by S. toxins, designated SLT-I and SLT-II. SLT-I is essentially dysenteriaetype 1 or EHEC is the progressionof the disease identicalto Shigatoxin, and its cytotoxic activityfor Vero or to the hemolytic-uremicsyndrome (HUS), which is charac- HeLa cells can be neutralizedwith anti-Shigaantibodies [4, terized by acute renal failure, thromboticmicroangiopathy, 5]. SLT-II is 567chomologous to Shiga toxin/SLT-I at the and thrombocytopenia[15]. Histopathologicstudies of the deduced amino acid level, and its cytotoxicityis not blocked kidneys of HUS patientshave shown profoundalterations in by anti-Shiga toxin antibodies [5, 6]. the glomeruli.More specifically,glomerular endothelial cells Shiga toxin and SLTs are holotoxins that consist of a sin- (EC) appearswollen, and there is abundantfibrin deposition gle 32- to 33-kDa A subunit in associationwith a pentamer and inflammatorycell influx in the lumina of the glomeruli of 7.7-kDa B subunits [7]. The A subunit is a specific N-gly- [16]. These observations,as well as the criticalrole of EC in cosidase that selectively cleaves a single adenine residue maintainingnormal blood flow [ 17], have led to the concept that systemic Shiga toxin or SLTs may specificallytarget the glomerularEC for damageand therebycontribute to the de- of HUS. In of this et al. Received 16 revised 13 1991. velopment support hypothesis,Obrig August 1990; February showed that toxin are for The opinions or assertionsherein are the privateones of the authorsand [18] Shiga preparations cytotoxic are not to be construedas official or reflectingthe views of the Department human umbilical vein EC (HUVEC) in vitro. of Defense or of the Uniformed ServicesUniversity of the Health Sciences. In the here, we assessed the Grant National Institutesof Health experiments reported cyto- support: (AI-20148). toxic of crude toxin, SLT-II,and Reprintsor correspondence:Dr. Alison O'Brien,Department of Microbi- potential Shiga affinity-pu- ology, Uniformed Services University of the Health Sciences, 4301 Jones rified Shiga toxin for confluent human saphenous vein EC BridgeRd., Bethesda,MD 20814. (HSVEC) and HUVEC monolayers. In addition, we com- * Presentaddress: Biocarb, Inc., Gaithersburg,Maryland. pared Shiga toxin-mediated EC and Vero cell cytotoxicity The Journalof InfectiousDiseases 1991;164:344-52 and examined the role of and re- This articleis in the publicdomain. lipopolysaccharides(LPS) 0022-1899/91/6403-0017 combinanthuman tumornecrosis factor-a in directEC cyto- JID 1991;164 (August) ShigaToxin and SLT-II-MediatedEC Toxicity 345 toxicity. Finally, we analyzed toxin binding to intact EC and IL). The stock preparationswere then diluted in growth medium quantitated levels of toxin receptor in EC membranes. as needed. Tissue culture. Vero and HeLa cells were maintained in Ea- gle's MEM (Flow Laboratories, McLean, VA) supplemented with \07c fetal bovine serum, 10 mM L-glutamine, 50 units/ml Materials and Methods penicillin, and 50 Mg/mlstreptomycin. EC were harvested from adult human saphenous veins (HSV) obtained during coronary Bacterial strains and S. 1 strain plasmids. dysenteriae type bypass operations as previously described [26]. Vein segments 3818T levels of toxin and was isolated from produces high Shiga (~2.5 cm) were transported to the laboratory in chilled blood a with severe in Central America E. coli patient dysentery [19]. with 100 units/ml preservative-freeheparin (Sigma). The HSV 0157:H7 strain 933 both SLT-I and II and was iso- - produces were cannulated and flushed with cold Ca"^ and Mg^ -free lated from an outbreak of colitis in the United hemorrhagic Dulbecco's PBS (PBS-CMF; GIBCO, Grand Island, NY). HSV States [2, Toxins used in this were derived from E. coli 5]. study were ligated after filling to slight distension with 0.17c CLS II DH5a Research Laboratories, (Bethesda Gaithersburg, MD) Freehold, transformed with recombinant the cloned collagenase (Worthington Diagnostic Systems, NJ) plasmids containing and 0.5^ bovine serum albumin (BSA) in PBS-CMF and im- toxin genes. E. coli harbors the plasmid that (pLPSH3) pBR328 mersed in Hanks' balanced salt solution (GIBCO) for 15 min at contains the entire Shiga toxin operon on a 5.9-kb Bglll-Sall 370C. HSV were flushed with 10 ml of cold complete medium insert subcloned from pNAS 10 [4,20]. E. coli (pLP32) harborsa (CM) composed of Medium 199 (GIBCO) with 207cfetal bovine Bluescribe vector (pBS[?] phagemid kit; Stratagene, La Jolla, serum (Hyclone Laboratories, Logan, UT), 100 pg/ml heparin CA) with a 3.0-kb Sphl-Kpnl fragment from pNN76 encoding (Sigma), 100 pg/ml L-glutamine, 50 units/ml penicillin, 50 pg/ SLT-II [21, 22]. Clones producing high levels of toxin were ml streptomycin (GIBCO), and 2 pg/ml amphotericin B (Fungi- maintained under BL3 -f- EK1 containment [23]. zone; GIBCO). Toxin preparations. LPS were prepared from S. dysenteriae The vein contents were centrifuged (200 g, 1 min, 40C), and 3818T and E. coli 933 by the hot aqueous-phenol method of the cell pellet was resuspended in 1.0 ml of CM Westphal and Jann [24]. The pooled aqueous phases were dia- supplemented with 20 /d/ml EC-mitogen extract prepared from bovine retina lyzed against nine changes of double-distilled, deionized HjO, using a modification of a described method concentrated 10-fold (Amicon, Danvers, MA), and lyophilized. previously [27, 28]. The cells were placed in wells of 24-well (Falcon; LPS preparationscontained
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