Binding and Transcytosis of Botulinum Neurotoxin by Polarized Human Colon Carcinoma Cells

Abstract

T-84 and Caco-2 human colon carcinoma cells and Madin-Darby canine kidney (MDCK) cells were used to study binding and transcytosis of iodinated Clostridium botulinum neurotoxin serotypes A, B, and C, as well as tetanus toxin. Specific binding and transcytosis were demonstrated for serotypes A and B in intestinal cells. Using serotype A as an example, the rate of transcytosis by T-84 cells was determined in both apical to basolateral (11.34 fmol/h/cm2) as well as basolateral to apical (8.98 fmol/h/cm2) directions, and by Caco-2 cells in the apical to basolateral (8.42 fmol/h/cm2) direction. Serotype A retained intact di-chain structure during transit through T-84 or Caco-2 cells, and when released on the basolateral side was toxicin vivo to mice and in vitro on mouse phrenic nerve-hemidiaphragm preparations. Serotype C and tetanus toxin did not bind effectively to T-84 cells, nor were they efficiently transcytosed (8–10% of serotype A). MDCK cells did not bind or efficiently transcytose (0.32 fmol/h/cm2) botulinum toxin. Further characterization demonstrated that the rate of transcytosis for serotype A in T-84 cells was increased 66% when vesicle sorting was disrupted by 5 μm brefeldin A, decreased 42% when microtubules were disrupted by 10 μm nocodazole, and decreased 74% at 18 °C. Drugs that antagonize toxin action at the nerve terminal, such as bafilomycin A1 (which prevents acidification of endosomes) and methylamine HCl (which neutralizes acidification of endosomes), produced only a modest inhibitory effect on the rate of transcytosis (17–22%). These results may provide an explanation for the mechanism by which botulinum toxin escapes the human gastrointestinal tract, and they may also explain why specific serotypes cause human disease and others do not. T-84 and Caco-2 human colon carcinoma cells and Madin-Darby canine kidney (MDCK) cells were used to study binding and transcytosis of iodinated Clostridium botulinum neurotoxin serotypes A, B, and C, as well as tetanus toxin. Specific binding and transcytosis were demonstrated for serotypes A and B in intestinal cells. Using serotype A as an example, the rate of transcytosis by T-84 cells was determined in both apical to basolateral (11.34 fmol/h/cm2) as well as basolateral to apical (8.98 fmol/h/cm2) directions, and by Caco-2 cells in the apical to basolateral (8.42 fmol/h/cm2) direction. Serotype A retained intact di-chain structure during transit through T-84 or Caco-2 cells, and when released on the basolateral side was toxicin vivo to mice and in vitro on mouse phrenic nerve-hemidiaphragm preparations. Serotype C and tetanus toxin did not bind effectively to T-84 cells, nor were they efficiently transcytosed (8–10% of serotype A). MDCK cells did not bind or efficiently transcytose (0.32 fmol/h/cm2) botulinum toxin. Further characterization demonstrated that the rate of transcytosis for serotype A in T-84 cells was increased 66% when vesicle sorting was disrupted by 5 μm brefeldin A, decreased 42% when microtubules were disrupted by 10 μm nocodazole, and decreased 74% at 18 °C. Drugs that antagonize toxin action at the nerve terminal, such as bafilomycin A1 (which prevents acidification of endosomes) and methylamine HCl (which neutralizes acidification of endosomes), produced only a modest inhibitory effect on the rate of transcytosis (17–22%). These results may provide an explanation for the mechanism by which botulinum toxin escapes the human gastrointestinal tract, and they may also explain why specific serotypes cause human disease and others do not. Botulinum neurotoxin (BoNT), 1The abbreviations used are: BoNT/Abotulinum neurotoxin serotype ACaco-2human colon adenocarcinomaMDCKMadin-Darby canine kidneyT-84human colon carcinoma. which is the etiologic agent responsible for the disease botulism, can enter the body by several different routes, but the most common of these is the gastrointestinal system (1Sanders A.B. Seifert S. Kobernick M. J. Fam. Pract. 1983; 16: 987-1000PubMed Google Scholar, 3Hatheway C.L. Curr. Top. Microbiol. Immunol. 1995; 195: 55-75Crossref PubMed Scopus (160) Google Scholar, 4Montecucco C. Schiavo G. Rossetto O. Arch. Toxicol. 1996; 18: 342-354Crossref Scopus (52) Google Scholar). Thus, most cases of intoxication are due to ingestion of food contaminated with preformed toxin or ingestion of food contaminated with bacteria that can produce toxin in the gut. In either case, BoNT escapes the gastrointestinal system to reach the general circulation (lymph and blood). Toxin in the blood is then distributed to peripheral cholinergic nerve endings, which are the target cells for toxin action. botulinum neurotoxin serotype A human colon adenocarcinoma Madin-Darby canine kidney human colon carcinoma. There is now a substantial literature describing the cellular, subcellular, and even molecular aspects of toxin action on cholinergic cells (5Montecucco C. Schiavo G. Mol. Microbiol. 1994; 13: 1-8Crossref PubMed Scopus (496) Google Scholar, 6Jahn R. Hanson P.I. Otto H. Ahnert H.G. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 329-335Crossref PubMed Scopus (29) Google Scholar, 7Oguma K. Fujinaga Y. Inoue K. Microbiol. Immunol. 1995; 39: 161-168Crossref PubMed Scopus (130) Google Scholar, 8Rossetto O. Deloye F. Poulain B. Pellizzari R. Schiavo G. Montecucco C. J. Physiol. ( Paris ). 1995; 89: 43-50Crossref PubMed Scopus (44) Google Scholar, 9Schiavo G. Rossetto O. Tonello F. Montecucco C. Curr. Top. Microbiol. Immunol. 1995; 195: 257-274PubMed Google Scholar). This literature derives from a model that envisions the toxin proceeding through three major steps (2, 10Simpson L.L. J. Pharmacol. Exp. Ther. 1980; 212: 16-21PubMed Google Scholar, 11Simpson L.L. Pharmacol. Rev. 1981; 33: 155-188PubMed Google Scholar, 12Simpson L.L. Annu. Rev. Pharmacol. Toxicol. 1986; 26: 427-453Crossref PubMed Scopus (397) Google Scholar, 13Montecucco C. Papini E. Schiavo G. FEBS Lett. 1994; 346: 92-98Crossref PubMed Scopus (216) Google Scholar). During the first step, the toxin binds to receptors that are highly localized in the junctional region of cholinergic cells. The toxin molecule, which is composed of a heavy chain (Mr ∼100,000) and a light chain (Mr ∼50,000) linked by a disulfide bond, is thought to rely on the carboxyl terminus of the heavy chain to associate with cells. During the second step, the toxin is productively internalized by vulnerable cells. This process is actually a sequence of two events: 1) penetration of the plasma membrane by receptor-mediated endocytosis, and 2) penetration of the endosome membrane by pH-induced translocation. Although there is much that remains to be determined about productive internalization, there is suggestive evidence that the amino terminus of the heavy chain plays an important role in this process. During the third and final step in toxin action, the light chain that reaches the cytosol acts as a zinc-dependent endoprotease to cleave polypeptides that are essential for exocytosis (6Jahn R. Hanson P.I. Otto H. Ahnert H.G. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 329-335Crossref PubMed Scopus (29) Google Scholar, 8Rossetto O. Deloye F. Poulain B. Pellizzari R. Schiavo G. Montecucco C. J. Physiol. ( Paris ). 1995; 89: 43-50Crossref PubMed Scopus (44) Google Scholar, 9Schiavo G. Rossetto O. Tonello F. Montecucco C. Curr. Top. Microbiol. Immunol. 1995; 195: 257-274PubMed Google Scholar). Blockade of exocytosis leads to the failure of neuromuscular transmission that is characteristic of botulism. In contrast to our knowledge of events at the nerve ending, very little is known about the cellular and subcellular events that account for the ability of the toxin to cross membranes in the gastrointestinal system (14Lamanna C. Hillowalla R.A. Alling C.C. J. Infect. Dis. 1967; 117: 327-331Crossref PubMed Scopus (15) Google Scholar, 15Sugii S. Ohishi I. Sakaguchi G. Infect. Immun. 1977; 17: 491-496Crossref PubMed Google Scholar, 16Bonventre P.F. Rev. Infect. Dis. 1979; 1: 663-667Crossref PubMed Scopus (38) Google Scholar). Those data that are available suggest that the mouth is the least effective site of absorption, the stomach is intermediate, and the intestine is the most effective site of absorption. There are no studies that actually demonstrate a mechanism for botulinum toxin penetration of gut cells. However, there is one self-evident matter that can be deduced. The series of steps that underlie toxin movement at the level of individual gut cells cannot be identical to the steps that underlie toxin movement in individual nerve cells. If BoNT were to enter and remain inside cells in the gastrointestinal system, this would prevent it from reaching peripheral cholinergic nerve endings. Thus, there must be fundamental differences that distinguish toxin movement across gastrointestinal membranes and movement across neuronal membranes. There are two broad categories of toxin transport that could account for movement from the gastrointestinal system to the general circulation. As by P.F. Rev. Infect. Dis. 1979; 1: 663-667Crossref PubMed Scopus (38) Google transport could a receptor-mediated mechanism or a In the to the that BoNT the gut by the process of specific binding and to this there be little or no pH-induced of toxin across membranes. this that toxin on the side of cells must be to the side of cells, and the transcytosed toxin must be in a that can cholinergic This by the and of BoNT A to a human of gut The data provide the first the cellular and subcellular events that account for toxin movement from the gastrointestinal system to the general circulation. T-84 human colon carcinoma cells, Caco-2 human colon carcinoma cells, and Madin-Darby canine kidney cells were from The and were from and were from were from and were from were from toxin was from A of was by of of were by Y. of and were to in the literature G. Pharmacol. Ther. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, L.L. PubMed Scopus Google Scholar). mice which were from were in an for of and to food and were and by the and T-84 cells were in a of and with and were at in T-84 cells were and when through were used for in this Caco-2 cells were in with and were at in Caco-2 cells were and when through were used for in this MDCK cells were in with and were at in MDCK cells were and when through were used for in this for membrane and to cells, the rate of of from the to the was determined as K. J. Biol. PubMed Scopus Google Scholar). to this the in both The was by of to the for from the to the were determined for or for that be by of cells with were on that with for 18 As an for membrane was for at the of this was to the but not the The was to the at and evidence of in the was for of when there was the data from these were not in the final B, and C, and tetanus as well as such as the were iodinated to The was in to the of of the The were to an specific of with a of L.L. J. Pharmacol. Exp. Ther. Google Scholar, L.L. J. Pharmacol. Exp. 1994; Google Scholar, J. L.L. J. Pharmacol. Exp. Ther. Google Scholar). neurotoxin in was to iodinated and on for The was by of of in for 5 The and were a that was with The toxin was with and were of was for The toxin which at was and at °C. Toxin in the was determined at the PubMed Scopus Google Scholar). A of this was in a to the of and were used to specific were and to as were three and then in with 10 and The cells were to °C. The in the well was with with or a of toxin. The cells were at for to to reach which cells and membranes were with 10 with were then of the and in a least three were of botulinum neurotoxin cells were in on membranes with a μm The was to with cells, the were with 10 I. was in and at °C. The was in as and 10 of were to The were to at the were light for by a with The was to of cells and Caco-2 or were at the with of in the and in the In these in the the apical of cells, and in the the basolateral of cells. was The were to for a of 10 of The of was by the rate of movement from the the were on that were 10 and The transcytosis was by botulinum neurotoxin either to the or of toxin was in two the of the were and on of a of was of as a of by to a final of in the were on The were three with and the of was in a were by identical at 10 °C. transport was by of the from the from was through a and were The of in the was determined in a toxin at and the in the was to the of toxin the two the was due to A of two was in for of toxin were by was to the at for The were to 18 a which the cells were and with or were at for 10 with then with μm 10 μm 5 μm A, μm cells were at in a The was an in a were at for 5 The was at for 10 The second was used for of toxin. for were on to PubMed Scopus Google or were for in on and to The of transcytosed that was from the was either by mice or by to mouse phrenic nerve-hemidiaphragm preparations. the in vivo transcytosed toxin or toxin was in a of with were for the of the the in vitro were and in that was with and at °C. The the and were and was was as a in to both in vivo and in vitro it was to the of toxin that was and This was in two the to be was to to the toxin The specific of the to the of the was The of in the from the from the and the specific were used to the of toxin. the of transcytosis as determined in this study were used to the of the toxin in the that were of transcytosis in of was in or MDCK was by of to the or were for at the the of were and were for and the toxin was to The of transcytosis was on the specific of toxin. The are for 18 Caco-2 are in a was in or MDCK was by of to the or were for at the the of were and were for and the toxin was to The of transcytosis was on the specific of toxin. The are for 18 Caco-2 are data from three or are as The of was the were either a for with or a for with were both to the of in and to the rate of movement of across these this was of The in three that the rate of from the apical to the side of T-84 cells the of The were for Caco-2 and MDCK These with by others K. J. Biol. PubMed Scopus Google and they are a that and molecular was were on that with for 18 to toxin did not in either T-84 or MDCK serotypes of botulinum toxin and tetanus toxin were and the ability of these to bind to intact cells was As in there was substantial and binding of serotypes A and B to T-84 cells. In the binding of serotype C and tetanus toxin was A series of was with MDCK cells, in which binding of only serotype A was there was no evidence of toxin binding to these cells were to the ability of T-84 cells to transcytose botulinum neurotoxin A, B, and C, as well as tetanus toxin. were on MDCK cells, which the ability to and but which not in toxin action. which data for serotype A, that T-84 cells are of and of toxin. In MDCK cells did not transcytose serotype A at a T-84 cells were also for ability to transcytose serotypes of botulinum toxin and tetanus toxin. the cells a substantial ability to the Botulinum toxin B was efficiently transcytosed of serotype C nor tetanus toxin (8–10% of was efficiently that T-84 cells transcytose The in that in toxin were with in transcytosis in T-84 cells. The of apical basolateral transcytosis increased from at to at toxin. the of toxin to the well in that was across cells decreased from at to at in T-84 were on MDCK cells, there was much of a for demonstrate that human cells transcytose serotype A was as a and was to rate In an was to apical basolateral transcytosis with basolateral apical The data on rate are in T-84 cells were to transcytose iodinated at a rate of when toxin was to the from the basolateral to the apical was (8.98 to 18 the rate of transcytosis to As in transcytosis of in Caco-2 cells was that in T-84 cells, but In transcytosis of the toxin by MDCK cells was and were used to the process of toxin movement across cells. and was on the that studies to or transcytosis I. J. PubMed Scopus Google Scholar, K. K. J. PubMed Scopus Google Scholar, S. J. PubMed Scopus Google Scholar, M. J. Biol. 1994; PubMed Scopus Google Scholar, Y. ( Y. ). 1994; PubMed Scopus Google Scholar). the results of the studies with brefeldin A, which to vesicle nocodazole, which to and of was increased by of cells with 5 μm brefeldin A, the rate of transcytosis was decreased by with 10 μm In with transcytosis of the toxin was decreased when was from to 18 °C. A1 and methylamine HCl toxin action at the nerve by pH-induced of the from to the cytosol K. J. Biol. PubMed Scopus Google Scholar, L.L. J. Pharmacol. Exp. Ther. 1983; Google L.L. J. Pharmacol. Exp. Ther. Google Scholar). these were for on toxin movement across T-84 cells. data from transcytosis with bafilomycin A1 or methylamine and in the of either of the in a in the rate of transcytosis bafilomycin A1 nor methylamine HCl toxin transport across gut of bafilomycin A1 or methylamine HCl on rate was with or bafilomycin A1 or methylamine were with bafilomycin μm final or methylamine HCl final for 18 to of was were in the of and transcytosis was for 18 The for T-84 are of for are in a was with or bafilomycin A1 or methylamine were with bafilomycin μm final or methylamine HCl final for 18 to of was were in the of and transcytosis was for 18 The for T-84 are of for are The that intestinal cells are of and of this not two important is the toxin that is transcytosed by T-84 or Caco-2 cells is the toxin that is across cells and on the side in a that can The to the first was by then the transcytosis and of this in T-84 and Caco-2 cells. The toxin was to to cells, in a from cells, and in the transcytosis across cells. The results the of both the and that which was by cells are in A and B. and the toxin from which toxin was and and the toxin that was iodinated for transcytosis of iodinated that was to the for transcytosis studies is in is an from the of T-84 cells. and are of the by these cells to the that the heavy chain and light chain of the toxin from the cytosol and from the basolateral side of T-84 cells were from to the cells. that transcytosed toxin is not during transit In B, an of iodinated toxin that was and to the basolateral side of Caco-2 cells. the heavy and light chain of the toxin by the cells were from to cells. that toxin to the basolateral side of cells retained the ability to neuromuscular two of were in vivo with vitro with mouse phrenic nerve-hemidiaphragm preparations. in vivo were with an of from the well of T-84 that were to and for for This of to of as on the of also was in and of the in this in an of This is to that when of toxin was used not of from the of T-84 cells to was not to at nor was an of from the well of MDCK to at The results in the are in with on binding and transcytosis data for in vitro that from the of T-84 to toxin that was to an neuromuscular of transcytosed toxin to of on the of were to a of The was This is to when of toxin was to Thus, of data with transcytosed toxin to data for of toxin demonstrated that transcytosed toxin retained Botulinum toxin A as of a in which it is with two major a of and and a that with neurotoxin were to these enter cells or are across cells The were iodinated to then to the T-84 cells. The of and were the as used in with toxin The which are in two major did in the cytosol of T-84 cells. The of this to be the intact that was to the there were that may there was little of to the The two major that were from the well were the and a of toxin that was in the These data suggest that the but little Botulinum toxin is an that a of food known as botulism. produce the toxin must through two and essential of toxin must the gastrointestinal system and be the general circulation. toxin must to peripheral cholinergic nerve endings, it is internalized to produce of The that the toxin is that both of these of events must with a of There is now a literature to binding and of BoNT at the cholinergic nerve This is in contrast to the literature on toxin transport in the for which there is a of on cellular, subcellular, and molecular the study was with the of to an mechanism by which intact and toxin could from the to the side of gut cells. were on two human of intestinal and as well as a canine of The results of this three 1) human gut cells can bind and transport 2) the that are transcytosed are the that are with human due to and the ability to bind and transcytose BoNT is not a of and cells that There are several in vivo that BoNT could to enter the general circulation and reach the peripheral there could be binding and by intestinal cells. there could be binding to cells of R. Rev. 1996; PubMed Google to the system, and the the toxin could cells or Infect. Immun. 1995; PubMed Google Scholar, Infect. Immun. PubMed Google which would then penetration of the gut. The in this study was to the of binding and transcytosis by cells, but it did not the of by cells. serotypes that were and there was no evidence for of of cellular membranes or was across and T-84 cells. such as cells did not to a as by the study of In cells that the movement of from one to the was that is in molecular and in J. Biol. PubMed Google and that T-84 junctional are to with 5 K. J. Biol. PubMed Scopus Google one can that of BoNT is to be There were several that the that movement of toxin across T-84 cells was an process. was of binding to T-84 cells, and this binding was in the of a of toxin. This that the transport process with a specific binding movement of toxin was when cells were at which that the transport process was movement of toxin was by that to such as brefeldin A and these data for the that human intestinal cells can bind and transcytose in vivo and in vitro that toxin to the side of cells was intact and Thus, when toxin was to the of the and of were from the of the produced in mice that was identical to that produced by toxin. of from the to in vitro with phrenic nerve-hemidiaphragm to of neuromuscular These data from were in with results from in which toxin from the basolateral side of cells to toxin. The data that human intestinal cells can bind and transcytose toxin in a that is and is to that the literature a of studies that demonstrate transcytosis of intact across intestinal cells R. J. Biol. PubMed Scopus Google Scholar, R. G. PubMed Scopus Google Scholar, K. J. Immunol. 1994; PubMed Scopus (29) Google Scholar, R. M. E. Infect. Immun. 1996; PubMed Google Scholar, J. Exp. PubMed Scopus Google and in cases the studies of transcytosis Y. ( Y. ). 1994; PubMed Scopus Google Scholar, S. M. B. J. Physiol. Google Scholar, R. F. F. M. Biol. 1995; PubMed Scopus Google Scholar, J. 1996; PubMed Scopus Google Scholar). The rate of transcytosis of determined in the study is the of that for but it is at the of the In this may to be that the rate of transcytosis of toxin is that of iodinated toxin. B. and is to or of There is highly suggestive evidence that the of BoNT transcytosis in this is to by the toxin. studies on human that are with human by ingestion of toxin and others are not and tetanus is that this is the one in the ability of human intestinal cells to bind and transcytose and were across T-84 and Caco-2 cells, but and tetanus toxin were at much This of that the of and tetanus toxin is due to the of these to from the gut to the general circulation. The results with are In a the of several botulinum were on and human neuromuscular J. L.L. J. Pharmacol. Exp. Ther. Google Scholar). This demonstrated that human are to the neuromuscular of on sequence and of human that this was a for J. L.L. J. Pharmacol. Exp. Ther. Google Scholar, R. A.B. L.L. ( ). 1995; PubMed Scopus Google Scholar). the results of the study on human are with the results of the study on human intestinal cells, they to the that the of an transcytosis mechanism in the gut may be to explain the of human disease due to is important to that movement of toxin across intestinal cells was different from movement in MDCK cells or cholinergic nerve cells. In to MDCK cells, there was no evidence for binding and cells that are not of toxin. In to cholinergic cells, that nerve toxin by receptor-mediated Toxin then escapes by pH-induced translocation. Thus, such as bafilomycin which prevents acidification of and methylamine which neutralizes acidification of are of and toxin action L.L. J. Pharmacol. Exp. 1994; Google Scholar, L.L. J. Pharmacol. Exp. Ther. 1983; Google Scholar, L.L. J. Pharmacol. Exp. Ther. Google Scholar). of these a major effect on transcytosis of toxin in gut cells. also be of the that a was was not an that it was efficiently Thus, specific endocytosis, and transport across T-84 cells. In such as were by T-84 cells, but only a of this was In this study the first of transcytosis of of BoNT through a of intestinal cells that The results of this light on a mechanism by which toxin human gut cells, and may explain why are to toxin serotypes but to the that BoNT binding and transcytosis could be to A.B. L.L. Infect. Immun. PubMed Google or be used as a for to this are and for on and of T-84 cells, for a of and in of and for during toxin