government site. This amino-acid variation is responsible for the striking difference in symptoms these two bacteria induce in a host, says Song. 2022 Nov 25;13:1016438. doi: 10.3389/fmicb.2022.1016438. With a size of 595 kDa, SiiE is the largest protein of the . In addition, similar to SNAP23-deficient cells, we observed an increased amount of typhoid toxin carrier intermediates in the cytosol of STX4-deficient cells infected with S. Typhi (although the difference did not reach statistical significance), most likely due to the failure of these carriers to fuse with the plasma membrane (Figure 6c, Figure 6figure supplement 1, and Figure 6figure supplement 2, and Figure 6source data 1). We infected cells with the resulting strains and examined the recruitment of CI-M6PR to the S. Typhi-containing vacuoles. doi: 10.1128/mbio.01916-21. The proteins playing a role in xocyticc pathway driving the export of typhoid toxin were also identified. (b) Western blot analysis of the expression of typhoid toxin in parental HEK293T and the SNAP-23-, VAMP7-, and STX4-deficient cells. It can also trigger overburdening. One remaining question not addressed in this article is whether CI-M6PR's role as a sorting receptor for typhoid toxin is shared across different host cells. Transcribed Image Text: There have been many cases of human infection with Salmonella caused by contact (or ingestion) with raw or undercooked chicken. Despite the vast difference in disease outcomes that S.Typhi and S.Typhimurium cause in humans, there are few genomic regions that are unique to S.Typhi. We have previously utilized such an approach for the study of the typhoid toxin incoming transport pathway (Chang et al., 2019). Normal, Key amino acid residues necessary for PltB binding to sugar moieties are also, S-CDT-mediated DNA damage primarily occurs, S-CDT-mediated DNA damage primarily occurs in the S and G 2 /M phases, Infection of HIEC-6 human intestinal epithelial cells with S. Javiana does not induce, Infection of C56BL/6 mice with WT S. Javiana results in a higher bacterial, MeSH Weinstein DL, O'Neill BL, Hone DM, Metcalf ES. Cells defective in SEC23B or SAR1B showed reduced levels of typhoid toxin carriers and toxin export to the extracellular space despite indistinguishable levels in the number of intracellular S. Typhi and the levels of typhoid toxin expression. Cells were infected with a S. Typhi strain expressing FLAG-tagged CdtB and the levels of fluorescence associated with typhoid toxin carriers were determined 24 hr after infection. PltA is an ADP-ribosyl transferase with an unknown cellular target, and CdtB is an atypical deoxyribonuclease, which inflicts DNA damage on the intoxicated cells. Values were fitted to an orthogonal polynomial regression of degree 2 to estimate the relationship between different dilutions and 50% of cells in the G2/M phase using R software version 3.4.4 (https://www.r-project.org). All cell lines were routinely tested for a mycoplasma by a standard PCR method. Salmonellosis is illness caused by the bacterium Salmonella. Taken together, these results indicate that differences in the effector protein composition dictates the ability of S. Typhi to intersect with the CI-M6PR sorting receptor and the subsequent packaging of typhoid toxin into vesicle carriers intermediates essential for its export to the extracellular space. Mutations accumulated through two pathways (anchored by protease mutations I50V or I84V). We found that in contrast to wild type, expression of SseJS151A had no effect in the recruitment of CI-M6PR to the S. Typhi-containing vacuole, and on the formation of typhoid toxin vesicle carrier intermediates (Figure 3e, Figure 3figure supplement 5 and Figure 3figure supplement 7, and Figure 3source data 1), indicating that the modification of the lipid composition of the SCV through SseJs catalytic activity influences CI-M6PR recruitment. Human intestinal epithelial Henle-407 cells (obtained from the Roy Curtiss III collection in 1987) and HEK293T cells (from the American Type Culture Collection) were cultured in DMEM supplemented with 10% fetal bovine serum. Cultured cells were grown onto six-well plates and infected with wild-type S. Typhi. Using this assay, we have found no evidence for the presence of extracellular proteases capable of degrading typhoid toxin (shown in the revised manuscript as Supplementary Figure S4). Douesnard-Malo F, Daigle F (2011) Increased persistence of Salmonella enterica serovar Typhi in the presence of Acanthamoeba castellanii. To validate this interaction, we infected cultured human epithelial cells with S. Typhi strains expressing FLAG-epitope-tagged versions of wild-type typhoid toxin or its PltBS35A mutant form and examined their interaction with CI-M6PR by immunoprecipitation and western blot analysis. It included detections of pathogens (e.g., Salmonella spp.) Detection and functionality of the CdtB, PltA, and PltB from Salmonella enterica serovar Javiana. In each case, we have analyzed at least two independently-generated mutant clones. Those severely at-risk clusters may benefit from personalized medicine and lifestyle intervention to improve their dysregulated metabolic traits, aiming to achieve healthier aging. Can the authors rule out its involvement in a separate step of the pathway? Relative toxin export was determined as indicated in (a). Cells defective in CLTC, AP3B1, or AP4M1 showed no defect in the formation of toxin carrier intermediates although AP4M1-deficient cells showed decreased toxin export (Figure 4bd, Figure 4figure supplement 2, Figure 4figure supplement 3, Figure 4figure supplement 4, and Figure 4source data 2). The results of an additional independent experiment are shown in Figure 4figure supplement 2. We found that in CIM6PR -/- cells, formation of the CdtB/Sec23 complex was impaired (these data are shown in a modified Figure 4). A two-part list of links to download the article, or parts of the article, in various formats. However, as in S. Typhi, NTS S-CDT influences the outcome of infection both in vitro and in vivoIMPORTANCE Nontyphoidal Salmonella (NTS) are a major cause of bacterial food-borne illness worldwide; however, our understanding of virulence mechanisms that determine the outcome and severity of nontyphoidal salmonellosis is incompletely understood. A unique aspect of typhoid toxin is that it is only produced when S. Typhi is located within mammalian cells (Fowler and Galn, 2018; Span et al., 2008). doi:10.1086/650733. Whereas other types of Salmonella bacteria cause salmonellosis or food poisoning, S. typhi is more toxic. The results of two additional experiments are shown in Figure 3figure supplement 1. n.s. ****: p<0.0001, unpaired two-sided t test. You should complement the CRISPR KO cell lines described in this study with a plasmid expressing the deleted gene to confirm that the phenotype is not due to a random mutation acquired during the process. Pathog Dis. To specifically examine the contribution of CI-M6PR to typhoid toxin sorting into vesicle carriers, we generated a CI-M6PR-deficient cell line by CRISPR/Cas9 genome editing (Figure 2a). In many countries, E. coli is not as common as Salmonella in causing foodborne illness and outbreaks, primarily due to inadequate surveillance measures. doi: 10.1111/cmi.12939. As a negative control we used a mutant version of typhoid toxin that carries a single amino acid substitution in its PltB subunit (PltBS35A). We are sorry to say that, after consultation with the reviewers, we have decided that this work needs a major revision prior to be be considered for publication by eLife. The relative toxicity was determined by the percentage of cells at the G2/M phase after treatment with the different of the infection media fitted by nonlinear regression. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Through an affinity purification approach, we identified CI-M6PR as the typhoid toxin packaging receptor, which we found to be robustly recruited to the S. Typhi-containing vacuole. The researchers took a deeper dive into understanding how those different amino acids affected the toxins behavior on a molecular level. More specifically we found that the plasma membrane SNARE proteins SNAP23 and syntaxin 4 are required for typhoid toxin transport to the extracellular space. I consider that the authors have addressed my and other reviewers' comments in a satisfactory manner, and this new version of the manuscript is greatly improved. Cells were then washed with DPBS three times, permeabilized with 0.1% Triton-100/DPBS for 20 min at room temperature, and then stained with the antibodies against CI-M6PR and GFP overnight at 4C, and Alexa 488 and 594-conjugated antibodies (Invitrogen) for 1 hr at room temperature. The toxin is secreted into the Salmonella-containing vacuole (SCV) of host cells, from where it must be transported to the extracellular space to carry out its action. -, den Bakker HC, Moreno Switt AI, Govoni G, Cummings CA, Ranieri ML, Degoricija L, Hoelzer K, Rodriguez-Rivera LD, Brown S, Bolchacova E, Furtado MR, Wiedmann M. 2011. Deng L, Song J, Gao X, Wang J, Yu H, Chen X, Varki N, Naito-Matsui Y, Galn JE, Varki A. mBio. The quantification of the levels of typhoid toxin in the infection media was carried out by serial dilutions as indicated in the legend for Figure 2. Qiu YF, Nambiar RB, Xu XB, Weng ST, Pan H, Zheng KC, Yue M. Front Microbiol. S. Paratyphi bacteria cause a similar, but milder illness, which comes under the same title. Toxigenicity. (e) Western blot analysis of the expression of typhoid toxin in parental HEK293T and Rip11-deficient cells carried out as indicated in (b). Can the authors discuss the relevance of an increased relative toxin export as seen after knockdown of AP3B1 and STX-11? (c) Toxicity of typhoid toxin in parental HEK293T and CI-M6PR-deficient cells. We found a marked reduction in the levels of fluorescent puncta associated with toxin carriers in SAR1B-deficient cells infected with S. Typhi despite similar expression levels of typhoid toxin (Figure 4e and f, Figure 4figure supplement 2 and Figure 4source data 2). The SNAP23/STX4 complex has been shown to interact with the v-SNARE VAMP-7 (Rhl et al., 2019; Williams et al., 2014), and we found here that cells defective in VAMP-7 showed reduced levels of typhoid toxin in the extracellular media. 2008;21(5):531538. FOIA being sick (vomiting) stomach cramps. 2016), is this mutant defective in recruiting CI-M6PR to the vacuole? Among the 2,400 Salmonella serotypes, three infect pigs: Salmonella choleraesuis, Salmonella typhimurium and Salmonella derby and among those only Salmonella typhimurium commonly causes clinical signs in humans. This was a curious finding since it was well established by complementary studies using Salmonella Typhimurium that CI-M6PR does not colocalize with the SCV. The transfected cells were selected in culture medium containing puromycin for 2 days, and isolated clones were screened by PCR genotyping to identify knockout cells. Agent: Salmonella typhi; Agent: Shiga toxin (Verocytotoxin)-Producing Escherichia coli; Agent: Shigella; Agent: Treponema pallidum; . An official website of the United States government. Protease mutations I50V or I84V ) a ) approach for the striking difference in symptoms these two bacteria in! Recruiting CI-M6PR to the vacuole established by complementary studies using Salmonella Typhimurium that CI-M6PR does not colocalize the! Membrane SNARE proteins SNAP23 and syntaxin 4 are required for typhoid toxin incoming transport pathway ( Chang al.... Persistence of Salmonella enterica serovar Typhi in the presence of Acanthamoeba castellanii seen after knockdown of and. Of the CdtB, PltA, and PltB from Salmonella enterica serovar Javiana, SiiE is the largest of. 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