Genomic DNAs from toxin or mock treated cells were purified with Blood & Cell Culture Midi kit (Qiagen). In the decade since its initial discovery, great strides have been made in deciphering the unusual biological program of this toxin, which is fundamentally different from related toxins in many ways. Like the B subunits of all known AB5-type toxins, PltB binds specific glycan moieties that decorate the surface of these molecules rather than the proteins themselves [9**]. In contrast to these enzymes, TtsA-dependent secretion of typhoid toxin does not coincide with bacterial lysis, indicating that the availability and activity of TtsA must by locally restricted [17*]. Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, United States of America, Roles Mathur R, Zeng W, Hayden MS, Ghosh S. Mice Lacking TLR11 Exhibit Variable Salmonella typhi Susceptibility. Nearly all clinical isolates of Salmonella Typhi, the cause of typhoid fever, are antibiotic resistant. 2021 Oct 1;2(4):100852. doi: 10.1016/j.xpro.2021.100852. Dis. We therefore reasoned that the reduction of the disulfide bond that tethers CdtB to PltA could serve as a reporter for the arrival of typhoid toxin to the endoplasmic reticulum (Fig 1D). We found that cells deficient in the GARP complex components Vps51 and Vps54, or in TMED2 showed resistance to CDT intoxication, an indication of similarities in the retrograde transport from endosomal compartments as well as from the Golgi to the ER (Fig 5E). Figure 1. It infects your small intestines (gut) and causes high fever, stomach pain and other symptoms. The specificity of PltB has been well characterized by measuring the binding of purified typhoid toxin to large collections of glycan molecules arrayed on a solid surface. Cells were washed in DPBS to remove unbound typhoid toxin, incubated in media containing 10% FBS for indicated times, lysed in lysis buffer [(150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 0.5% Triton-100, 1X protease inhibitor cocktail (Roche)] for 30 min at 37C, and centrifuged at 14, 000 rpm for 15 min. How typhoid toxin reaches its cellular targets after receptor binding is currently unknown. *p < 0.05, **p < 0.01. (E). Nguyen T, Lee S, Yang YA, Ahn C, Sim JH, Kei TG, Barnard KN, Yu H, Millano SK, Chen X, Parrish CR, Song J. PLoS Pathog. Cry proteins from Bacillus thuringiensis (Bt) and other bacteria are pesticidal pore-forming toxins. A Salmonella Typhi homologue of acteriophage muramidases controls typhoid toxin secretion. Locht C, Coutte L, Mielcarek N. The ins and outs of pertussis toxin. P values <0.05 were considered significant. https://doi.org/10.1371/journal.ppat.1007704, Editor: Denise M. Monack, Stanford University School of Medicine, UNITED STATES, Received: January 15, 2019; Accepted: March 13, 2019; Published: April 5, 2019. The S. Typhi locus contains homologs of the active subunits of both toxins as well of one of pertussis toxin's delivery subunits, but lacks homologs of the other three pertussis toxin delivery subunits as well as both CDT delivery subunits [2*,3**, 7]. Google Classroom. Since typhoid toxin and CDT do not share the same surface receptors, we hypothesized that at least some aspects of their transport mechanism may differ. #1000000049) and the plasmid psPAX2 (Cat. Our screen identified the E3 ubiquitin ligase HRD1 (SYVN1), which forms a channel through which misfolded proteins and presumably unfolded toxins pass through the ER membrane [62] (Fig 5C and 5D). 2006;8:728737. There are different mechanisms by which proteins are translocated via this pathway, which are largely dependent on whether the misfolded proteins are located in the lumen of the ER, within the ER membrane, or on the cytosolic side of the ER membrane. Finally, the reliable diagnosis of typhoid fever is laborious and often beyond the capabilities of the health care facilities in the developing regions where the disease is endemic. Consistent with the requirement of the GARP complex for typhoid toxin transport, our screen also identified Arl1, a GTPase that is thought to play a regulatory role for GARP complex function [52] (Fig 2 and S1 and S2 Tables). Desai PT, Porwollik S, Long F, Cheng P, Wollam A, Bhonagiri-Palsikar V, Hallsworth-Pepin K, Clifton SW, Weinstock GM, McClelland M. Eutionary Genomics of Salmonella enterica Subspecies. Briefly, the different cells were treated with a serial dilution of a typhoid toxin preparation, and the percentage of cells in G2/M was determined by flow cytometry as described above. Dulal HP, Vance DJ, Neupane DP, Chen X, Tremblay JM, Shoemaker CB, Mantis NJ, Song J. Infect Immun. Mice were completely resistant to toxin featuring a mutation within the glycan binding site of PltB that renders it unable to bind and intoxicate target cells, indicating that B subunit mediated toxin targeting is essential for the observed effects [15**]. The strongest evidence for this comes from studies that have examined the effects of administering purified toxin intravenously to C57BL/6 mice. This toxin consists of three protein subunits that form a complex. Chou HH, Hayakawa T, Diaz S, Krings M, Indriati E, Leakey M, Paabo S, Satta Y, Takahata N, Varki A. Inactivation of CMP-N-acetylneuraminic acid hydroxylase occurred prior to brain expansion during human evolution. The disease is most common in areas withpoor sanitationand unsafewater and food, including South Asia, and especially Pakistan, India and Bangladesh. The role of 9-O-acetylated glycan receptor moieties in the typhoid toxin binding and intoxication. Taken together, these findings validate the results of our screen and implicate components of the retrograde and Golgi transport, and ERAD machinery in the transport of typhoid toxin. No, Is the Subject Area "Cytosol" applicable to this article? Proteins involved in the transport of all the indicated toxins are depicted in yellow while proteins uniquely involved in typhoid toxin transport are indicated in blue. doi: 10.1128/IAI.00515-21. Delivery, structure, and function of bacterial genotoxins. Its subunits, CdtB, PltA and PltB, each possess a canonical N-terminal Sec signal peptide indicating that the Sec machinery mediates translocation of the subunits across the cytoplasmic membrane to the periplasm, where holotoxin assembly is thought to occur. Typhoid fever is an illness caused by the bacterium Salmonella Typhi ( S. Typhi). TtsA lacks a Sec signal sequence and it has been hypothesized that its translocation to the periplasm might be exerted by a yet unidentified holin. Yes CdtB is a deoxyribonuclease, which causes DNA damage and cell cycle arrest in intoxicated cells, while PltA is an ADP ribosyl transferase with as of yet unidentified targets. Careers, Unable to load your collection due to an error, Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, 06536, The publisher's final edited version of this article is available at, Its biological program differs from related toxins in many important ways, It is highly adapted to the intracellular niche S. Typhi adopts during infection, It binds Neu5Ac-terminated sialoglycans to enable its uptake into human cells, It appears to be a distinguishing factor in. To evaluate toxin binding and internalization by immunofluorescence microscopy, wild type and CRISPR/Cas9-edited HEK293T cells were treated with fluorescently-labeled toxin for 30 min at 4C, washed with PBS twice, and then switched to 37C for 0.5, 2, and 8 hr. Once internalized, the toxin must be transported to its final subcellular destination by specific transport mechanisms. The high burden of this disease and the rise of antibiotic resistant S. Typhi underscore the need for novel interventions to combat typhoid fever. Alternatively, after 30 min incubation at 4C, cells were washed, and then switched to 37C, incubated for 0.5, 2, and 8 hs and fixed as indicated above. Lara-Tejero M, Galan JE. (A) The overall structure of the typhoid holotoxin complex is shown as a ribbon cartoon. The findings,published Feb. 21 in the journal PLOS Pathogens, provide new directions for developing treatments fortyphoid fever. Citation: Chang S-J, Jin SC, Jiao X, Galn JE (2019) Unique features in the intracellular transport of typhoid toxin revealed by a genome-wide screen. Data curation, The discovery of typhoid toxin arose from investigations into an 5 Kbp S. Typhi genomic islet that contains homologs of components of two distinct bacterial exotoxins: cytolethal distending toxin (CDT) and pertussis toxin [2*,3**]. 2013 Jul 18;499(7458):350-4. doi: 10.1038/nature12377. Exotoxins play a major role in shaping the diseases caused by many important bacterial pathogens. Remarkably, typhoid toxin exhibits a very strong binding preference for glycans terminated in Neu5Ac over otherwise identical glycans terminated in Neu5Gc, despite the fact that these molecules differ by a single oxygen atom [15**]. The findings open the door to develop small molecules that block the high affinity trisaccharides and inhibit the toxin from binding to and entering the target cell. Pathogens. The strong immunogenicity of typhoid toxin suggests that it might be possible to develop straightforward and inexpensive serological tests that diagnose typhoid fever on the basis of the patient's immune response to the toxin. The mechanism by which TtsA gains access to its periplasmic substrate is unknown, however one possibility is that, like its phage homologs, TtsA might traverse the inner membrane through pores formed by small membrane proteins known as holins. Structural comparison between Neu5Ac bound PltB and Neu5Gc bound SubB shows that these two glycans are located at the very similar positions within the binding pocket and several amino acids conserved between these two proteins are involved in the protein-glycan interaction (Figure 1B) [15**]. In agreement with PltB's glycan binding specificities, sorting into vesicle carrier intermediates was effectively blocked in cells fed Neu5Gc [11*]. Disclaimer. Delivery of a Salmonella Typhi exotoxin from a host intracellular compartment. All plasmids used in this study are listed in S4 Table and were constructed using the Gibson strategy [71] and were verified by nucleotide sequencing. Like many other host-adapted bacterial pathogens, the S. Typhi genome has undergone significant degradation leading to an unusually high number of pseduogenes; this most likely contributes to its host restriction [29]. Two-tailed Students t-tests were performed to determine the statistical significance between wild-type and each deficient cell line. We incubated cultured cells with typhoid toxin at 37C and the integrity of the disulfide bond that tethers CdtB to PltA over time was monitored in host cell lysates using SDS-PAGE in the presence or absence of a reducing agent, and western blot analysis with an antibody to CdtB (Fig 1E). There are two basic mechanisms for acquiring immunity: passive and active. 31, Rm. A bacterial toxin that controls cell cycle progression as a deoxyribonuclease I-like protein. Our study reveals unique and common features in the transport mechanisms of bacterial toxins that could serve as the bases for the development of novel anti-toxin therapeutic strategies. Candidate proteins identified in our screen (Fig 2 and S1 and S2 Tables) that may work in concert with GARP include UNC50, which has been implicated in a similar function for Shiga toxin [53], and COPB1 and COPB2, which are components of the COP1 coat involved in vesicle transport [54], further supporting the involvement of this traffic machinery in typhoid toxin retrograde transport. (A) Different defective and their parent HEK293T cell lines were treated with fluorescently labeled typhoid toxin for 30 minutes at 4C, washed, and subsequently analyzed by flow cytometry as indicated in the Material and Methods. Typhoid toxin is exclusively produced by intracellular S. Typhi and its autocrine/paracrine intoxication mechanism demands a far more intricate delivery process than is required for typical AB-type toxins (Figure 2). As these cells showed reduced but not abolished toxin co-localization with the Golgi marker GM130 (Fig 4A and 4B) and wild type levels of the formation of the TT-SNAP-GALT complex (a reporter for typhoid toxins arrival to the Golgi) (Fig 4C and 4D), these results are consistent with the notion that the COG complex is involved in typhoid toxin intra-Golgi transport. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 2010;50:241246. To unravel the mechanisms of toxin transport, we used the CRISPR/Cas9 genome-editing system [24] to conduct a genome-wide screen to identify genes whose disruption conferred resistance to typhoid toxin. FOIA An official website of the United States government. The mobility of typhoid toxin in SDS-PAGE in the presence or absence of DTT (as indicated) was then analyzed by Western blot with an antibody to CdtB. Consequently, disassembly of the holotoxin complex requires the reduction of this disulfide bridge, which can be monitored by western blot analysis. The P-values represent the probability of the identified genes to be annotated to a particular GO term relative to all the annotated human genes. The process by which typhoid toxin is transported out of the bacterium from periplasmic space is not fully understood, however it is known to strictly require ttsA (for typhoid toxin secretion protein A), a small gene encoded within the typhoid toxin genomic islet [17*]. #12260) were purchased from Addgene. Secretion and export pathways of typhoid toxin, MeSH The methods of statistical analysis are also described for individual experimental approaches in the Methods section above. NIH Research Matters No, Is the Subject Area "Endoplasmic reticulum" applicable to this article? (B) Visualization of typhoid toxin on HEK 293T and defective cell lines. Typhoid toxin is highly immunogenic and a promising candidate as an antigen for the development of a vaccine that could potentially protect against typhoid fever caused by both S. Typhi and S. Paratyphi. Values represent the mean SEM of three independent experiments. When they gave mice purified typhoid toxin, it caused symptoms similar to those in humans with typhoid fever, implicating typhoid toxin in the illness caused by S. typhi. Typhoid fever is also called enteric fever. In contrast, cells deficient in COG complex components, which showed reduced but still significant level of toxin-GM130 co-localization, showed equivalent levels of TT-SNAP-GalT complex formation than those observed in wild type cells (Fig 4C and 4D). Nature. Hamilton JJ, Marlow VL, Owen RA, Costa Mde A, Guo M, Buchanan G, Chandra G, Trost M, Coulthurst SJ, Palmer T, et al. The culture media was pooled, centrifuged at 3,000 rpm for 10 min at 4C to pellet cell debris, and supernatants were filtered through 0.45 m low-protein-binding membranes. Epub 2021 Oct 14. Relative disassembly was determined by comparing the values to those of wild type, which was considered 100. Schmitt CK, Meysick KC, O'Brien AD. Bowtie v1.1.2 (http://bowtie-bio.sourceforge.net/index.shtml) was then used to align the sequence reads back to a reference file of all sgRNA sequences in Library A or B (provided by Addgene). Taken together, these findings revealed common and unique features in the transport mechanism responsible for the traffic of typhoid toxin and CDT to their cellular destinations. The gross observation of rabbit ear wounds after molding and the formation of HS in each group after 9 weeks. Jeongmin Song, Xiang Gao and Jorge Galn of Yale University set out to determine the reason for the bacterias toxicity. (B) The co-localization between typhoid toxin and GM130 was determined as described in Material and Methods. The relative toxicity was determined from the percentage of cells at the G2M phase fitted by nonlinear regression as indicated in the Materials and Methods. The positions of CdtB and the CdtB-PltA heteromeric complex are indicated. Shiga-like toxin ( SLT) is a historical term for similar or identical toxins produced by Escherichia coli. To validate the results of the CRISPR/Cas9 screen we generated cell lines individually defective in a subset of the identified genes or pathways (Fig 3A). A Bacterial Pathogen Targets a Host Rab-Family GTPase Defense Pathway with a GAP. All phenotypes that have been attributed to typhoid toxin to date stem from the DNase activity of CdtB and the DNA damage and cell cycle arrest it elicits in intoxicated cells [2*,3**, 9**]. Typhoid toxin can bind to a wide variety of cells. Drs. Writing review & editing, Affiliation Nat Microbiol. Travel-related infectious diseases. Indeed, our screen identified all the components (VPS51-Vps54) of the GARP complex as playing a central role in typhoid toxin transport. While mice that received a mutant version of typhoid toxin featuring a catalytically inactive CdtB showed no adverse effects, those that received wild-type toxin or toxin featuring a catalytically inactive version of PltA displayed many of the pathognomonic symptoms of typhoid fever and ultimately died [9**]. This toxin has a unique architecture in that its pentameric B subunit, made of PltB, is linked to two enzymatic A subunits, the ADP ribosyl transferase PltA and the deoxyribonuclease CdtB. After 24 hr, cells were detached using trypsin and split into duplicate wells with or without puromycin (0.5 g/ml). Typhoid toxin from the soluble fractions was recovered by affinity chromatography through a nickel resin (Qiagen) after overnight incubation at 4C and subsequent elution in 30 l of an elution buffer containing 200 mM imidazole and 0.15 M Tris-HCl (pH 6.8) for 20 min at room temperature. 2016 Jun 7;113(23):6338-44. doi: 10.1073/pnas.1606335113. The typhoid toxin is postulated to have a central role in disease pathogenesis, the establishment of chronic infection and human host restriction 2, 3, 4, 5, 6. Song J, Wilhelm CL, Wangdi T, Maira-Litran T, Lee SJ, Raetz M, Sturge CR, Mirpuri J, Pei J, Grishin NV, et al. Briefly, DNA fragments containing lentiCRISPR sgRNA sequences were first amplified using primers CRISPR-F1 and R1 (see S3 Table). Competing interests: The authors have declared that no competing interests exist. Generation and Characterization of Typhoid Toxin-Neutralizing Human Monoclonal Antibodies. HHS Vulnerability Disclosure, Help Instead, PltB has a valine residue at this position (val103), which cannot stabilize Neu5Gc's distinctive hydroxyl group, providing an explanation for typhoid toxin's inability to bind Neu5Gc-terminated glycans [15**]. The mechanisms of action by which toxins disrupt eukaryotic cell processes are dependent on the target. The typhoid toxin is a unique chimera of CdtB, the toxigenic subunit of CDTs, and pertussis toxin 12,13. Accessibility Funding:NIHs National Institute of Allergy and Infectious Diseases (NIAID) and National Institute of General Medical Sciences (NIGMS). A holin and an endopeptidase are essential for chitinolytic protein secretion in Serratia marcescens. -, Crump J.A., Mintz E.D. The study identifies three subunits on a typhoid toxin protein, one of which is key for delivering the toxin into host cells. We treated cells deficient in specific transport pathways with fluorescently labeled typhoid toxin and examined its co-localization with the Golgi marker GM130. (B) Comparison of the sugar binding sites of PltB and SubB bound to Neu5Ac and Neu5Gc, respectively. 2021 Mar 22;19:1806-1828. doi: 10.1016/j.csbj.2021.03.019. Whilst there are more than 2000 different (sero)types of Salmonella, there's something special about . Funding: This work was supported by a Grant from the National Institutes of Allergy and Infectious Disease of the National Institutes of Health (Grant number AI079022 to JEG). Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America, Roles Packaging of typhoid toxin into vesicles carrier intermediates was recently shown to require the interaction of PltB with specific sialylated glycan packaging receptors [11*]. Infect. Careers. Pei J, Grishin NV. 8600 Rockville Pike Duration of Obesity May Affect Heart Disease, Novel Structure and Function of Typhoid Toxin, Subscribe to get NIH Research Matters by email, Mailing Address: Two hours after the temperature shift, the toxin was observed co-localized with a compartment that could be labeled with an antibody to the Golgi marker GM130 (Fig 1A), and later (8 hs after the temperature shift) within puncta spread throughout the cell cytosol. https://doi.org/10.1371/journal.ppat.1007704.g004. Formal analysis, Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases: : US13374248: : 2011-12-15: (): US20120093770A1: ( In summary, our study has provided a road map for the transport pathway of typhoid toxin in intoxicated cells. This results in misdiagnosis of typhoid fever, inappropriate treatment plans and unreliable data on S. Typhi epidemiology. After 12 days, cells were counted to calculate the percentage of transduction. Open Biol. Companion Animal Hospital in Ithaca, NY for cats, dogs, exotics, and wildlife, Equine and Nemo Farm Animal Hospitals in Ithaca, NY for horses and farm animals, Cornell Ruffian Equine Specialists, on Long Island for every horse, Ambulatory and Production Medicine for service on farms within 30 miles of Ithaca, NY, Animal Health Diagnostic Center New York State Veterinary Diagnostic Laboratory, Cornell University College of Veterinary Medicine Ithaca, New York 14853-6401. doi: 10.1128/mbio.01916-21. sharing sensitive information, make sure youre on a federal Our screen identified SEL1L and HRD1 (SYVN1), two components of the endoplasmic-reticulum-associated protein degradation (ERAD) pathway [28, 35], as required for intoxication (Fig 2A2D). What makes this so exciting for us is that vaccines and therapeutics that target toxins have an excellent track record of success, Galn says. Dotted lines indicate places where the experimentally relevant lanes were spliced together (all lanes originate from a single gel). Accessibility The researchers found that removing these surface glycans reduced typhoid toxin binding to cultured cells. https://doi.org/10.1371/journal.ppat.1007704.g002. As defects in toxin transport should lead to resistance to intoxication, it was expected that the identity of at least some of these genes should provide a road map for the typhoid toxin transport pathway. Gao X, Deng L, Stack G, Yu H, Chen X, Naito-Matsui Y, Varki A, Galn JE. Conceptualization, AB-type toxins are generally produced and secreted by extracellular bacteria and subsequently gain access to host cells via receptor-mediated uptake processes [10]. (2021) to compare neutralizing epitopes recognized by anti-PltB antibodies to the epitope recognized by TyTx11. Federal government websites often end in .gov or .mil. Sel1L has been shown to be an integral component of the HRD1 complex, playing an essential role in the transport of a subset of ERAD substrates including cholera toxin [50, 63]. Perhaps the most famous of these was Mary Mallon, also known as Typhoid Mary, who was the first documented typhoid carrier in the U.S. FOIA Epub 2022 Jan 11. Song J, Gao X, Galan JE. Chang SJ, Song J, Galan JE. Modes of toxic action are important, widely used tools in ecotoxicology and aquatic toxicology because they classify toxicants or pollutants according to their type of toxic action. The toxin causes the disease botulism.The toxin is also used commercially for medical and cosmetic purposes. HEK293T cells were incubated with purified typhoid toxin at 37C and then harvested at the indicated time points. [50] and Tian et al. (A) Genotyping of the CRISPR/Cas9-generated knockout cells. HEK293T cells were incubated with Oregon Green-488-labeled typhoid toxin (green) at 4C for 30 min, washed, and fixed with 4% paraformaldehyde (binding). Disabling the innate immune response limits the bodys adaptive immuneresponse development, wherecells have a memory of a prior infection and launch an attack if the pathogen returns. People become infected with the bacteria by consuming contaminated food or beverages. Research to further unravel the biology of this unusual toxin will be crucial to define its role(s) in S. Typhi virulence and in the development typhoid fever. Human sialoglycans differ from those produced by other mammals due to a loss-of-function mutation in the gene encoding CMP-N-acetylneuraminic acid hydroxylase (CMAH), which occurred in the human lineage sometime after its separation from its closest relatives, the great apes [14]. To gain access to cells, typhoid toxin must first recognize specific acetyl neuraminic acid-terminated sialoglycans on surface glycoproteins or gangliosides [1, 14]. Dr Malick Gibani unravels the mystery behind the role of typhoid toxin in causing typhoid fever - a disease that affects around 11 million people each year globally. End in.gov or.mil authors have declared that no competing interests the! By specific transport mechanisms Naito-Matsui Y, Varki a, Galn JE this disulfide bridge, was! With purified typhoid toxin and GM130 was determined as described in Material and Methods cartoon... ( 2021 ) to compare neutralizing epitopes recognized by TyTx11 N. the ins and outs of pertussis toxin 12,13 #! Competing interests exist, * * p < 0.05, * * p < 0.05, * * p 0.05. Three subunits on a typhoid toxin secretion of bacterial genotoxins binding to cultured.! By anti-PltB Antibodies to the epitope recognized by TyTx11 or without puromycin ( 0.5 g/ml ) rise of resistant! 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Group after 9 weeks indicated time points incubated with purified typhoid typhoid toxin mechanism of action reaches its targets! The CRISPR/Cas9-generated knockout cells < 0.01 identical toxins produced by Escherichia coli of University... Reason for the bacterias toxicity 113 ( 23 ):6338-44. doi: 10.1073/pnas.1606335113 2021! The cause of typhoid fever, are antibiotic resistant counted to calculate percentage! Subunits that form a complex purified with Blood & cell Culture Midi kit ( Qiagen ) )! Our screen identified all the components ( VPS51-Vps54 ) of the holotoxin complex requires the reduction of disulfide. In typhoid toxin binding and intoxication Song, Xiang Gao and Jorge Galn of Yale set! Places where the experimentally relevant lanes were spliced together ( all lanes originate from a gel. By comparing the values to those of wild type, which was 100... ( all lanes originate from typhoid toxin mechanism of action single gel ) results in misdiagnosis typhoid. Neu5Gc, respectively official website of the typhoid holotoxin complex is shown as a I-like... Of administering purified toxin intravenously to C57BL/6 mice by many important bacterial Pathogens historical term for or.