Salmonella Typhi, the cause of typhoid fever in humans. This toxin has an unusual biology in that it is produced by Salmonella Typhi only when located within host cells. Once synthesized, the toxin is secreted to the lumen of the Salmonella-containing vacuole from where it is transported to the extracellular space by vesicle carrier intermediates. Here, we report the identification of the typhoid toxin sorting receptor and components of the cellular machinery that packages the toxin into vesicle carriers, and exports it to the extracellular space. We found that the cation-independent mannose-6-phosphate receptor serves as typhoid toxin sorting receptor and that the coat protein COPII and the GTPase Sar1 mediate its packaging into vesicle carriers. Formation of the typhoid toxin carriers requires the specific environment of the Salmonella Typhi-containing vacuole, which is determined by the activities of specific effectors of its type III protein secretion systems. We also found that Rab11B and its interacting protein Rip11 control the intracellular transport of the typhoid toxin carriers, and the SNARE proteins VAMP7, SNAP23, and Syntaxin 4 their fusion to the plasma membrane. Typhoid toxin’s cooption of specific cellular machinery for its transport to the extracellular space illustrates the remarkable adaptation of an exotoxin to exert its function in the context of an intracellular pathogen.Typhoid toxin is an essential virulence factor for  Salmonella enterica serovar Typhi is the cause of typhoid fever in humans, a systemic disease that affects approximately 20 million people every year resulting in approximately 150,000 deaths worldwide  and is increasingly becoming more prevalent in certain areas of the world. Both serovars are restricted to the human host and no animal reservoir for these pathogens has been identified. The emergence of multiple-antibiotic resistant strains of these pathogens, which are becoming endemic in different parts of the world, is a major concern as it is complicating the available therapies   linked to a pentameric ‘B’ subunit alternatively composed of PltB or PltC -terminated sialoglycans, which are almost unique to the human host since most other mammals display glycolyl neuraminic acid (Neu5Gc)-terminated glycans  . Homologerovars) , althougerovars) . Typhoid or PltC . PltA is glycans . A humannfection . Howevernfection , aspectsnfection .S. Typhi is located within mammalian cells  through a novel dedicated protein secretion system . This mutant cannot be packaged into vesicle carrier intermediates because this residue is critical for its interaction with its putative Neu5Ac-bearing sorting receptor  analysis . As a nereceptor . The catreceptor  was iden control . To vali version . Consistnfection . Recruit vacuole . These ohimurium , which dS. Typhi to gain access to and replicate within these cells  and non-typhoidal (e.g. S. Typhimurium) S. enterica serovars. For example, while the Rab-family GTPases Rab29, Rab32, and Rab38 are robustly recruited to the S. Typhi-containing vacuole, these GTPases are absent in the vacuolar compartment harboring S. Typhimurium  encoded by S. Typhimurium but absent from S. Typhi may preclude the recruitment of CI-M6PR to the SCV and thus prevent the packaging of typhoid toxin into transport carriers. To test this hypothesis, we individually expressed in S. Typhi the S. Typhimuirum SPI-1 or SPI-2 T3SS effector proteins that are absent or pseudogenes in S. Typhi  in S. Typhi and examined the recruitment of CI-M6PR to the S. Typhi-containing vacuole. 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  expressing FLAG epitope-tagged CdtB. The formation of a typhoid toxin/Sec23 complex was then investigated by co-immunoprecipitation. We found that typhoid toxin could be detected in complex with SEC23 in HEK293T parental cells infected with wild-type S. Typhi  also known as Rip11 . This eftal cell , despitetal cell  and typhtal cell , a phenotal cell . Taken tN-ethylmaleimide-sensitive factor-activating protein receptors (SNAREs), which mediate the fusion between the carrier vesicle and target membranes , most likely due to the failure of these carriers to fuse with the plasma membrane (SNAP23 has been shown to cooperate with other SNARE proteins such as syntaxin 4 (STX4) and syntaxin 11 (STX11) to mediate membrane fusion . Therefoell line  despite ell line . In contell line . In addimembrane .The SNAP23/STX4 complex has been shown to interact with the v-SNARE VAMP-7 . TherefoS. Typhi-containing vacuole, is packaged into vesicle intermediates, and then transported to the extracellular milieu  recruits Rab32 and Rab38, which are involved in melanosome biogenesis  were purchased from the indicated commercial sources.Antibodies to CI-M6PR , anti-FLAG M2 , and to S. Typhi strains were diluted 1/20 in LB broth medium containing 0.3 M NaCl and grown until they reached an OD600 of 0.9. Culture cells were infected as described before (S. Typhi or S. Typhimurium strains at a multiplicity of infections (MOI) of 30 or 10, respectively, in Hank’s balanced salt solution, and then incubated with culture medium containing 100 µg/ml gentamicin for 1 hr to kill extracellular bacteria. Cells were washed and incubated for the indicated times with medium containing 10 µg/ml gentamicin to avoid cycles of reinfection. All cell lines were routinely tested for a mycoplasma by a standard PCR method.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. Overnight cultures of the d before . BrieflyS. Typhi strains expressing FLAG-epitope-tagged CdtB, cells were fixed in 4% paraformaldehyde and then blocked with 3% BSA, 0.3% Triton X-100 in DPBS. Fixed cells were incubated with primary mouse monoclonal anti-FLAG M2 (Sigma) and rabbit polyclonal anti-S. Typhi lipopolysaccharide (Sifin) antibodies followed by Alexa 488-conjugated anti-mouse and Alexa 594-conjugated anti-rabbit antibodies (Invitrogen). Samples were visualized under a Nikon TE2000 fluorescence microscope equipped with and Andor Zyla 5.5 CMOS camera driven by Micromanager software (https://www.micro-manager.org). Quantification of typhoid toxin export carrier intermediates has been previously described (https://imagej.nih.gov/). The LPS stain was used to identify the area corresponding to the bacterial cell body and this area was used to obtain the bacterial-associated typhoid toxin fluorescence signal, which was substracted from the typhoid toxin-associated fluorescence. The remaining fluorescence was defined as the area of typhoid toxin carrier intermediates. The intensity of fluorescence associated with toxin carriers was normalized using the fluorescence-associated typhoid toxin within bacterial cells in the same field.The visualization of typhoid toxin vesicle carrier intermediates was carried out as previously described . Brieflyescribed . BrieflyS. Typhi or S. Typhimurium strains expressing superfolder GFP were fixed with ice-cold methanol for 5 min at –20°C. 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 4°C, and Alexa 488 and 594-conjugated antibodies (Invitrogen) for 1 hr at room temperature. Cells were then observed under a Nikon TE2000 fluorescence microscope and images captured with an Andor Zyla 5.5 CMOS camera driven by Micromanager software (https://www.micro-manager.org). Co-localization between CI-M6PR and SCV were analyzed by performing the plug-in Manders’ overlap coefficient in ImageJ.Henle-407 cells infected with the different Addgene # 62988; http://n2t.net/addgene:62988). PX459 carrying sgRNA were transfected into HEK293T cells using Lipofectamine 2000. 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. At least two independently isolated clones per cell line were examined for the relevant phenotypes.CRISPR/Cas9 genome editing was carried out as described previously . The sgRS. Typhi within host cells  containing protease inhibitors for 30 min on ice, and then centrifuged for 15 min at 14,000×rpm at 4°C. The supernatants were incubated with 30 μg of the purified typhoid toxin overnight at 4°C. Next day, the protein complex was incubated with 20 μl of anti-FLAG M2 agarose for 2 hr at 4°C. Immunoprecipitation of protein complexes were collected by centrifugation at 500× escribed .6 onto 10 cm dishes. Twenty-four hours later, cells were infected with S. Typhi strains expressing FLAG-epitope-tagged CdtB or mock-infected for 1 hr at an MOI of 10. Infected cells were harvested at 24 hr post infection and lysed in lysis buffer  containing protease inhibitors . After 30 min on ice, samples were centrifuged for 15 min at 14,000×rpm at 4°C. The supernatants were incubated with 20 μl of anti-FLAG M2 agarose for 24 hr at 4°C. Protein complexes were collected by centrifugation at 500× g for 1 min, washed three times with lysis buffer, and then resuspended in 30 μl of Laemmli sample buffer. Samples were analyzed by SDS-PAGE and western blot with antibodies against FLAG and CI-M6PR.HEK293T cells were seeded at a density of 2 × 10S. Typhi. Twenty-four hours after infection, the supernatant from infected cells was collected and filtered through 0.2 μm syringe filters, diluted as indicated in each experiment, and applied to fresh wild-type HEK293T cells. The toxic effect of different dilutions of the culture supernatant was determined at 48 hr post treatment as previously described (https://www.r-project.org). Values analyzed from the R program were normalized relative to those of the parental cells, which were considered 100 and are the mean ± SD of three independent experiments.Cultured cells were grown onto six-well plates and infected with wild-type escribed . BrieflyS. Typhi (as listed in the Key resources table) were cloned into the pET28a (Novagen) expression vector. E. coli strains carrying the plasmids encoding the different toxins were grown at 37°C in LB media to an OD600 of ~0.6, toxin expression was induced by the addition of 0.5 mM IPTG, and cultures were further incubated at 25° C overnight. Bacterial cell pellets were resuspended in a buffer containing 15 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1 mg/ml DNase I, 0.1 mg/ml lysozyme, and 0.1% PMSF and lysed by passaging through a cell disruptor (Constant Systems Ltd.). Toxins were then purified from bacterial cell lysates through affinity chromatography on a Nickel-resin (Qiagen), ion exchange, and gel filtration (Superdex 200) chromatography as previously described ; p values below 0.05 were considered statistically significant.The following software was used in this study: GraphPad Prism (plotting data), Micro-Manager, Slidebook 6, Imaris 9.7 (Oxford Instruments), Adobe Illustrator & Adobe Photoshop (image preparation), FlowJo , and Image Studio Lite (Li-COR Biosciences), and Image Lab  (quantification of the band intensity of western blot). Salmonella Typhi-infected cells to the extracellular space including mannose-6-phosphate Receptor that serves sorting receptor.This paper is of interest to microbiologists as well as eukaryotic cell biologists interested in vesicular trafficking pathways. The authors identify several eukaryotic proteins required for typhoid toxin export from  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Decision letter after peer review:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]Salmonella Typhi infected cells" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Ethel Bayer-Santos (Reviewer #3).Thank you for submitting the paper "Typhoid toxin sorting and exocytic transport from Comments to the Authors:eLife.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 Despite being generally positive about the nature of this study, the reviewers have raised serious issues with this paper that they feel will take some time to address. While I am rejecting the paper as a result of this, I am broadly supportive of this manuscript and if you feel that you are able to address these issues, I will consider a newly submitted form of this paper that I will treat as a revised manuscript.Among the shortcomings identified, the four points listed below would need to be addressed experimentally:1. In order for you to conclude that COPII is required for SCV-plasma membrane trafficking they would have to show that 1) knockdown of sec23 does not have an indirect effect on M6PR by blocking its ER export – you could do this by examining the localization of M6PR at the TGN and endosomes by immunofluorescence microscopy and 2) COPII can mediate trafficking from the SCV You could do this by checking for co-localization of COPII components with the SCV or the typhoid toxin.2. You should control for the possibility that secreted toxin undergoes proteolysis in cells lacking M6PR (or other proteins). If this is the case, then important conclusions might be unjustified.3. 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. Alternatively, you could show same phenotype for independently generated mutants4. The interaction between CdtB and Sec23 should be tested as control in the m6pr knockout background.Reviewer #1:Salmonella Typhi, the causative agent of typhoid fever in humans. 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. In this manuscript by Chang et al. the authors investigate the pathway by which the typhoid toxin is trafficked out of the host cell. They use immunoprecipitation followed by mass spectrometry to identify the cation-independent mannose-6-phosphate (CI-M6PR) as a binding partner for the typhoid toxin and convincingly demonstrate its role as the typhoid toxin sorting receptor. In addition, they show that the Typhimurium effector SseJ blocks recruitment of CI-M6PR to the SCV, confirming that the expression of specific Type III secretion system effectors affects the environment of the SCV. They then use CRISPR/cas9 knockdowns to characterise other host trafficking proteins that are required for toxin export, including COPII, Sar1, Rab11B, Rip11, VAMP7, SNAP23 and Syntaxin 4. These data provide interesting insights into the requirements of host proteins for typhoid toxin export, but a single pathway is not convincingly established, as proteins identified have not been linked together in terms of interaction or dependency with respect to toxin secretion. In addition, the data need to be discussed in the context of established knowledge of eukaryotic trafficking pathways.The typhoid toxin is produced by -1, TIP47 and Rab9 and the specific steps of trafficking pathways that they are involved in. The authors should discuss how their data relates to these pathways. AP1 is known to interact with M6PR and to traffic to and from endosomes and should therefore be included in the screens or at least discussed with a clear reason for its omission. The authors should also comment on the fact that the CI-M6PR normally traffics from the TGN to both endosomes and the plasma membrane, which could provide a plausible pathway.1. The results of this work should be discussed in the context of the large body of published work on intracellular trafficking. M6PR intracellular trafficking pathways have been well characterised with information on the signalling sequences found in its cytoplasmic tail, the adaptor proteins they interact with including AP1, AP2, GGAs, PACS2. COPII mediates trafficking of proteins between the ER and the Golgi. As such its knockdown should block all export from the ER including that of M6PR. The authors should discuss their data in the context of this information. To be convincing the authors need to rule out any indirect effect caused by blocking ER export and demonstrate clearly that COPII mediates this atypical pathway. For example, it is important to know (1) if COPII is recruited to the SCV membrane in a toxin-dependent manner, (2) if COPII interacts with M6PR and (3) is such an interaction required for toxin export in a way that can be distinguished from general ER export?3. In order to convincingly establish a pathway, the proteins identified should be linked experimentally. For example, does the interaction of the toxin with Sec23 depend on the presence of M6PR? Can the authors be certain that all identified proteins are involved in direct traffic from the SCV to the plasma membrane? AP4 knockdown also decreased toxin export. Can the authors rule out its involvement in a separate step of the pathway?Salmonella infection and perturbation of mannose-6-phosphate receptor traffic have been shown to increase the secretion of proteases , which could degrade extracellular secreted toxin. Why do the authors use an indirect assay for cell cycle arrest rather than western blot of supernatants? Western blot analysis should be used to determine if lower amounts in extracellular fractions can be rescued by protease inhibition. Can the authors discuss the relevance of an increased relative toxin export as seen after knockdown of AP3B1 and STX-11?4. The relative toxin export assay is not a convincing measurement. 5. The screen of Typhimurium effectors is interesting – in relation to Typhimurium. But the results do not help understand the features of the Typhi SCV that result in CI-M6PR recruitment. Since the Typhi spiA mutant is defective in packaging toxin into transport carriers , is this mutant defective in recruiting CI-M6PR to the vacuole? Or is CI-M6PR recruitment simply part of a default phago-lysosomal pathway, as would occur with dead bacteria?Reviewer #2:Their data well justify the authors' conclusions. The article provides an excellent example highlighting the adaptation of an exotoxin co-evolved in the context of the life cycle of an intracellular pathogen producing the toxin. 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. This question arises because the interaction between CI-M6PR and typhoid toxin PltB is through the glycan, N-acetylneuraminic acid, displayed on the glycosylated sorting receptor(s), suggesting a different glycoprotein might serve as a sorting receptor in other host cells. Nonetheless, this manuscript elegantly determined the detailed sorting and transport process of typhoid toxin occurring in S. Typhi-infected host cells, which is well-justified by the data presented in the manuscript.1. 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. This question arises because the interaction between CI-M6PR and typhoid toxin PltB is through the glycan, N-acetylneuraminic acid, displayed on the glycosylated sorting receptor(s), suggesting a different glycoprotein might serve as a sorting receptor in other host cells. This can be addressed by investigating other cells relevant to S. Typhi infection. Alternatively, the authors can address this point in the Discussion, perhaps under a separate section "Limitations of this study" to help readers obtain a clearer understanding of the toxin trafficking pathway.2. SseJ's role is demonstrated, which is absent in S. Typhi. In some places in the manuscript , the corresponding descriptions are a bit confusing, sounding like a SPI2 effector produced by S. Typhi plays a key role in the packaging process. Rephrasing some of the descriptions may be considered to help readers get the concept quickly.3. Relating to SseJ's role, examining the altered expression and/or localization of OSBP and CI-M6PR on the SCV in various cases tested in the manuscript  is expected to strengthen the authors' point on the co-evolution of SPI2 effectors and typhoid toxin.4. S. Figures S1, S2, S4, S7, and S8: All data from 2-3 independent experiments can be combined and presented as the main figures .Reviewer #3:Salmonella Typhi typhoid toxin , which is responsible for sorting this toxin out of the Salmonella-containing vacuole (SCV). 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 authors went on to determine the reason for such differences and identified the S. Typhimurium-specific T3SS effector SseJ as responsible for preventing the co-localization of CI-M6PR in SCVs.In this manuscript by Chang et al., authors identified the intracellular receptor  of The proteins playing a role in the exocytic pathway driving the export of typhoid toxin were also identified. After binding of the typhoid toxin to CI-M6PR in the lumen of the SCV, COPII and SarIB mediate the formation of export carries, which are transported to the plasma membrane in a manner dependent on the GTPase Rab11B and its interacting partner RIP11. Lastly, the fusion of the carriers to the plasma membrane depends on the SNARE proteins VAMP7, SNAP23 and STX4.This work answers a long-standing question in the field, and the author conclusions are well supported by a series of complementary experiments.Some controls are missing in this version of the manuscript, which could provide strength to these findings.In my opinion, the work would greatly beneficiate from live-imaging microscopy analysis showing the trafficking of typhoid toxin within infected or transfected cells. These data could complement the current methodology and make the article more interesting.1) I think that it is important to complement all 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 . As all data was performed in HEK293T cells that transfect very well, this could be done relatively easily for CI-M6PR-/-; SEC23B-/-; Sar1B-/-; Rab11B-/-; Rip11-/-; SNAP23-/-; STX4-/-; VAMP7-/-.2) The co-IP experiment shown in Figure 4I could be performed in the CI-M6PR-/- background. If the authors hypothesis is correct, one would expect that the interaction between CdtB and Sec23 (COPII) would be lost.3) Although discussed in the text, Figure 6C is lacking statistical analysis. This should be included.Salmonella Typhi infected cells" for further consideration by eLife. Your revised article has been evaluated by Dominique Soldati-Favre (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Typhoid toxin sorting and exocytic transport from The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:They concur that the manuscript is significantly improved and recommended a few points to be addressed (that will not involve new experiments) prior to consideration for acceptance.Reviewer #1:In general, the authors have addressed the main concerns of the reviewers and the manuscript is much approved.As COPII is responsible for ER export there is a high possibility that blocking it through knockout could result in the mislocalisation of many proteins and subsequent off-target effects; this is not fully addressed.In addition, the authors do not discuss the extensive literature on M6PR trafficking including the role of AP1, nor do they conclusively rule out the role of AP1 in the trafficking of M6PR-bound toxin.These points could be mentioned in the 'limitations of this study' paragraph in the Discussion.Reviewer #2: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.Reviewer #3:The revised version of the manuscript has been significantly improved. The authors have addressed all my concerns raised in the initial review. [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]Among the shortcomings identified, the four points listed below would need to be addressed experimentally:1. In order for you to conclude that COPII is required for SCV-plasma membrane trafficking they would have to show that 1) knockdown of sec23 does not have an indirect effect on M6PR by blocking its ER export – you could do this by examining the localization of M6PR at the TGN and endosomes by immunofluorescence microscopy.In the original submission of the article, we stated that "Recruitment of the packaging receptor CI-M6PR to the S. Typhi-containing vacuoles in SEC23B-deficient cells was indistinguishable from the recruitment observed in the parental cells, indicating that the defect in typhoid toxin packaging was not due to the impaired recruitment of CI-M6PR . We assume the reviewer missed these data.2) COPII can mediate trafficking from the SCV You could do this by checking for co-localization of COPII components with the SCV or the typhoid toxin.Salmonella containing vacuole (SCV). We have found the recruitment of Sec23A to the SCV, particularly at later times after infection. This observation  is consistent with the involvement of COPII in the packaging and trafficking of typhoid toxin to the extracellular space.As requested by the Reviewer we have examined the co-localization of Sec23A to the 2. You should control for the possibility that secreted toxin undergoes proteolysis in cells lacking M6PR (or other proteins). If this is the case, then important conclusions might be unjustified.S. Typhi-infected CI-M6PR cells by exposing purified typhoid toxin to those supernatants. Using this assay, we have found no evidence for the presence of extracellular proteases capable of degrading typhoid toxin .We have assessed this question by examining for the presence of proteases that could potentially degrade typhoid toxin on cell supernatants of 3. 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. Alternatively, you could show same phenotype for independently generated mutantsIn each case, we have analyzed at least two independently-generated mutant clones. These data are shown in several additional Supplementary Figures.4. The interaction between CdtB and Sec23 should be tested as control in the m6pr knockout background.We have compared the ability of CdtB (a component of typhoid toxin) to form a complex with Sec23 in the presence or absence of CI-M6PR. We found that in CI-M6PR -/- cells, formation of the CdtB/Sec23 complex was abrogated. This observation further supports the premise that the CI-M6PR/COPII complex serves as cargo receptor for typhoid toxin .Reviewer #1:The typhoid toxin is produced by Salmonella Typhi, the causative agent of typhoid fever in humans. 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. In this manuscript by Chang et al. the authors investigate the pathway by which the typhoid toxin is trafficked out of the host cell. They use immunoprecipitation followed by mass spectrometry to identify the cation-independent mannose-6-phosphate (CI-M6PR) as a binding partner for the typhoid toxin and convincingly demonstrate its role as the typhoid toxin sorting receptor. In addition, they show that the Typhimurium effector SseJ blocks recruitment of CI-M6PR to the SCV, confirming that the expression of specific Type III secretion system effectors affects the environment of the SCV. They then use CRISPR/cas9 knockdowns to characterise other host trafficking proteins that are required for toxin export, including COPII, Sar1, Rab11B, Rip11, VAMP7, SNAP23 and Syntaxin 4. These data provide interesting insights into the requirements of host proteins for typhoid toxin export, but a single pathway is not convincingly established, as proteins identified have not been linked together in terms of interaction or dependency with respect to toxin secretion. In addition, the data need to be discussed in the context of established knowledge of eukaryotic trafficking pathways.We thank the reviewer for the positive comments and the very thorough review of our work. We also understand that the reviewer may feel that our data do not "convincingly establish…a single pathway" responsible for toxin export, and that the reviewer would also like to see our data "interpreted in the context of established knowledge of eukaryotic trafficking pathways". The view of the reviewer is, understandably, "basic cell biology-centric", a view fundamentally shaped by paradigms built through the study of "model systems" . It is important to note, however, that as those of us who have been in the field of "cellular microbiology" from the very early days like to say, "bugs do not read the cell biology textbooks". As the reviewer would certainly understand, this makes the work of studying pathogens much harder because, although knowledge of cell biological process available is extremely useful, often we can't fit our observations into preestablished canons. "Bugs" don't let us do that… There are countless examples of cell biological processes modulated by microbial pathogens in ways that are "unprecedented", and that deviate from established paradigms. This is actually one of the exciting aspects of the study of pathogens because "unprecedented" often times does not mean that bugs do it "their way" but rather, that the study of the canonical systems have missed that aspect of cell biology. In these cases, the studies not only illuminate microbial pathogenesis but also basic cell biology. This study falls somewhere in between… there are mechanistic aspects of typhoid toxin export that fit some previously established paradigm but there are aspects that clearly do not. Consequently, it is natural that the pathway does not neatly fit into a "single pathway". It is "the nature of the beast"… Regardless, when appropriate, we have tried to place our findings in the context of previously described pathways. Again, we thank the reviewer for the thorough review of our work.-1, TIP47 and Rab9 and the specific steps of trafficking pathways that they are involved in. The authors should discuss how their data relates to these pathways. AP1 is known to interact with M6PR and to traffic to and from endosomes and should therefore be included in the screens or at least discussed with a clear reason for its omission. The authors should also comment on the fact that the CI-M6PR normally traffics from the TGN to both endosomes and the plasma membrane, which could provide a plausible pathway.1. The results of this work should be discussed in the context of the large body of published work on intracellular trafficking. M6PR intracellular trafficking pathways have been well characterised with information on the signalling sequences found in its cytoplasmic tail, the adaptor proteins they interact with including AP1, AP2, GGAs, PACSIdeally, we would have preferred to identify components of the typhoid toxin export pathway through a “genome-wide” screen as we have done for the incoming endocytic pathway using CRISPR/Cas9 . Unfortunately, for a variety of reason, it is not possible to implement such screen for the study of typhoid toxin’s export pathway. Consequently, to identify components of typhoid’s toxin export pathway we conducted a more labor-intensive “gene candidate screen", which required the construction of more than 20 different knockout cells using CRISPR/Cas9, amounting to a major undertaking. Using this approach, it is practically impossible to probe every gene reported to be involved in some exocytic pathway. Therefore, we had to prioritize our study focusing on those genes that may be more likely to play a role in typhoid toxin export. For example, the identification of the CI-M6PR sorting receptor allowed us to narrow the set of candidates to investigate. Furthermore, we approached the study in a gradual manner so as to be able to use the information obtained from the examination of some candidates to select additional ones. As alluded by the reviewer, numerous adaptors and coat proteins have been shown to interact with CI-M6PR in different cellular compartments. Among them, and as indicated by the reviewer, are the adaptor proteins AP1 and AP2, which in the context of the sorting of CI-M6PR, have been shown to work in conjunction with clathrin. Since we found no involvement of clathrin in typhoid toxin sorting, we directed our attention to other adaptor/coat components known to be involved in the sorting of the CI-M6PR but in a clathrin-independent manner. We have clarified these issues in the text and expand our discussion of possible link between known CI-M6PR sorting pathways and typhoid toxin secretion.2. COPII mediates trafficking of proteins between the ER and the Golgi. As such its knockdown should block all export from the ER including that of M6PR. The authors should discuss their data in the context of this information. To be convincing the authors need to rule out any indirect effect caused by blocking ER export and demonstrate clearly that COPII mediates this atypical pathway. For example, it is important to know (1) if COPII is recruited to the SCV membrane in a toxin-dependent manner, (2) if COPII interacts with M6PR and (3) is such an interaction required for toxin export in a way that can be distinguished from general ER export?Salmonella containing vacuole (SCV), particularly at later times after infection. This observation  is consistent with the involvement of COPII in the packaging and trafficking of typhoid toxin to the extracellular space. The luminal localization of typhoid toxin, which does not have any transmembrane domain, makes it highly unlikely that it could be involved in the recruitment of CI-M6PR or COPII to the SCV. Therefore, we have not examined this specific question.As discussed above, we have shown that Sec23A is recruited to the 3. In order to convincingly establish a pathway, the proteins identified should be linked experimentally. For example, does the interaction of the toxin with Sec23 depend on the presence of M6PR? Can the authors be certain that all identified proteins are involved in direct traffic from the SCV to the plasma membrane? AP4 knockdown also decreased toxin export. Can the authors rule out its involvement in a separate step of the pathway?We examined whether the interaction between Sec23 and typhoid toxin is dependent on the presence of CI-M6PR . As discussed above, we found that in the absence of CI-M6PR, the formation of a complex between CdtB (a typhoid toxin component) and Sec23 was abrogated. This observation supports the notion that typhoid toxin, CI-M6PR and Sec23 form a complex. As indicated by the reviewer, the absence of AP4 resulted in a reduced level of typhoid toxin export. However, we did not observe a decrease in the formation of typhoid toxin transport carriers, suggesting a role for AP4 at some step of typhoid toxin export, downstream from its budding from the SCV, or an indirect effect on other aspect of typhoid toxin export. Whether the involvement of AP4 is direct or indirect is not clear at this point. The limitations of our study have now been specifically addressed in the Discussion.4. The relative toxin export assay is not a convincing measurement. Salmonella infection and perturbation of mannose-6-phosphate receptor traffic have been shown to increase the secretion of proteases , which could degrade extracellular secreted toxin. Why do the authors use an indirect assay for cell cycle arrest rather than western blot of supernatants? Western blot analysis should be used to determine if lower amounts in extracellular fractions can be rescued by protease inhibition. Can the authors discuss the relevance of an increased relative toxin export as seen after knockdown of AP3B1 and STX-11?Salmonella Typhimurium results in a redistribution of the CI-M6PR and the release of proteases to the infection supernatant. However, we stressed that the intracellular biology of Salmonella Typhi is substantially different from that of S. Typhimurium. For example, although the Rab GTPases Rab29, Rab32, and Rab38 are readily and robustly recruited to the S. Typhi-containing vacuole, they are not recruited to the S. Typhimurium-containing vacuole. This difference is specifically due to the absence in S. Typhi of two T3SS effector proteins (SopD2 and GtgE), which effectively prevent the recruitment of these GTPases to the S. Typhimurium-containing vacuole . More relevant to this study, we have shown here that in sharp contrast with S. Typhimurium containing vacuole, CI-M6PR is readily and robustly recruited to the S. Typhi-containing vacuole, although we did not observe the recruitment of lysosomal hydrolases (i. e. CI-M6PR recruitment was not due to the delivery of S. Typhi to lysosomes) . Therefore, observations made with the study of S. Typhimurium vesicle trafficking cannot be directly extrapolated to S. Typhi. In any case, we have specifically addressed the reviewer's concern and found no evidence for the presence of extracellular proteases capable of degrading typhoid toxin .We are aware that it has been reported that infection of cultured cells with The amount of toxin exported from infected cells is below the western blot detection limit under our experimental conditions. To be able to detect the toxin by western blot, the experiments would have to be scaled up in a manner that is not practical and susceptible to artifacts. Consequently, we had to use a more sensitive assay to detect typhoid toxin based on its biological activity. We are uncertain about the reasons why AP3B1- and STX-11-defective cells exhibit increased typhoid toxin export. We hypothesize that these proteins may be involved in alternative export pathways that may compete for cellular components involved in typhoid export but we do not have direct proof for this hypothesis.5. The screen of Typhimurium effectors is interesting – in relation to Typhimurium. But the results do not help understand the features of the Typhi SCV that result in CI-M6PR recruitment. Since the Typhi spiA mutant is defective in packaging toxin into transport carriers , is this mutant defective in recruiting CI-M6PR to the vacuole? Or is CI-M6PR recruitment simply part of a default phago-lysosomal pathway, as would occur with dead bacteria?S. Typhi ∆spiA mutant is directed to lysosomes. However, in this compartment, as we have previously shown, the mere presence of CI-M6PR on the SCV is not sufficient for typhoid toxin packaging into vesicle carrier intermediates since packaging requires the intracellular environment of the wild-type SCV (see PMID: 27832592). We understand that there is some confusion on this issue since historically the S. Typhi and S. Typhimurium vacuoles have been assumed to be similar. However, as we have discussed above, the SCVs in S. Typhi and S. Typhimurium infected cells are substantially different including differences in their ability to recruit the CI-M6PR, as well as substantial differences in the recruitment of at least 3 Rab GTPases. Stating the obvious, we would like to emphasize that the presence of the CI-M6PR on the wild-type S. Typhi SCV is not due to its traffic to lysosomes. Lysosomal hydrolases, for example, are not recruited to the wild-type S. Typhi containing vacuole . We have clarified this issue in the text and provided an additional Supplementary Figure to formally document this observation .The reviewer is correct and, as expected, the Reviewer #2:Their data well justify the authors' conclusions. The article provides an excellent example highlighting the adaptation of an exotoxin co-evolved in the context of the life cycle of an intracellular pathogen producing the toxin. 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. This question arises because the interaction between CI-M6PR and typhoid toxin PltB is through the glycan, N-acetylneuraminic acid, displayed on the glycosylated sorting receptor(s), suggesting a different glycoprotein might serve as a sorting receptor in other host cells. Nonetheless, this manuscript elegantly determined the detailed sorting and transport process of typhoid toxin occurring in S. Typhi-infected host cells, which is well-justified by the data presented in the manuscript.We thank the reviewer for the positive assessment of this work.1. 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. This question arises because the interaction between CI-M6PR and typhoid toxin PltB is through the glycan, N-acetylneuraminic acid, displayed on the glycosylated sorting receptor(s), suggesting a different glycoprotein might serve as a sorting receptor in other host cells. This can be addressed by investigating other cells relevant to S. Typhi infection. Alternatively, the authors can address this point in the Discussion, perhaps under a separate section "Limitations of this study" to help readers obtain a clearer understanding of the toxin trafficking pathway.The reviewer is correct, and it remains formally possible that typhoid toxin may engage alternative receptors in different cells. We have presented evidence that CI-M6PR appears to be the main receptor in intestinal Henle-407 and HEK293T cells, two rather different cell lines, which suggest that CI-M6PR is likely the main sorting receptor in several different cells, which is consistent with its ubiquitous expression. We have discussed this and other limitations of our study in the discussion session as suggested by the reviewer.2. SseJ's role is demonstrated, which is absent in S. Typhi. In some places in the manuscript , the corresponding descriptions are a bit confusing, sounding like a SPI2 effector produced by S. Typhi plays a key role in the packaging process. Rephrasing some of the descriptions may be considered to help readers get the concept quickly.We have introduced the suggested editorial changes.3. Relating to SseJ's role, examining the altered expression and/or localization of OSBP and CI-M6PR on the SCV in various cases tested in the manuscript  is expected to strengthen the authors' point on the co-evolution of SPI2 effectors and typhoid toxin.S. Typhi and S. Typhi expressing either wild-type SseJ or its catalytic mutant SseJS151A. We found that expression of wild type SseJ but not of its catalytic mutant prevented the efficient recruitment of CI-M6PR to the SCV. This led us to conclude that SseJ prevents the recruitment of CI-M6PR in a catalytic-dependent manner .Perhaps the reviewer missed the data, but we did compare the recruitment of CI-M6PR to the SCV of cells infected with wild-type 4. S. Figures S1, S2, S4, S7, and S8: All data from 2-3 independent experiments can be combined and presented as the main figures .We thank the reviewer for the suggestion. However, we prefer the format that we have used to show our data, which more directly demonstrate the reproducibility of the observations.Reviewer #3:In this manuscript by Chang et al., authors identified the intracellular receptor  of Salmonella Typhi typhoid toxin , which is responsible for sorting this toxin out of the Salmonella-containing vacuole (SCV). 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 authors went on to determine the reason for such differences and identified the S. Typhimurium-specific T3SS effector SseJ as responsible for preventing the co-localization of CI-M6PR in SCVs.The proteins playing a role in xocyticc pathway driving the export of typhoid toxin were also identified. After binding of the typhoid toxin to CI-M6PR in the lumen of the SCV, COPII and SarIB mediate the formation of export carries, which are transported to the plasma membrane in a manner dependent on the GTPase Rab11B and its interacting partner RIP11. Lastly, the fusion of the carriers to the plasma membrane depends on the SNARE proteins VAMP7, SNAP23 and STX4.This work answers a long-standing question in the field, and the author conclusions are well supported by a series of complementary experiments.Some controls are missing in this version of the manuscript, which could provide strength to these findings.In my opinion, the work would greatly beneficiate from live-imaging microscopy analysis showing the trafficking of typhoid toxin within infected or transfected cells. These data could complement the current methodology and make the article more interesting.We thank the reviewer for the positive assessment of our work. We share the reviewer’s desire to visualize the trafficking of typhoid toxin in live cells. Unfortunately, despite a lot of effort we have not been able to do so, fundamentally, because we can’t get fluorescently tagged versions of typhoid toxin to be efficiently secreted to the bacterial periplasm, despite testing many different tags, some of which reported in the literature to be transported to the bacterial periplasm. This has been an insurmountable challenge that has so far eluded a solution.1) I think that it is important to complement all 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 . As all data was performed in HEK293T cells that transfect very well, this could be done relatively easily for CI-M6PR-/-; SEC23B-/-; Sar1B-/-; Rab11B-/-; Rip11-/-; SNAP23-/-; STX4-/-; VAMP7-/-.In the revised version of the manuscript, we include the analysis of a least two independent clones of the CRISPR/Cas9 generated cell lines with deficiencies in different component of the export pathway . We found no significant differences in toxin trafficking between the independent clones.2) The co-IP experiment shown in Figure 4I could be performed in the CI-M6PR-/- background. If the authors hypothesis is correct, one would expect that the interaction between CdtB and Sec23 (COPII) would be lost.As suggested by the reviewer, we compared the ability of CdtB (a component of typhoid toxin) to form a complex with Sec23 in the presence or absence of CI-M6PR. We found that in CIM6PR -/- cells, formation of the CdtB/Sec23 complex was impaired . This observation further supports the premise that the CI-M6PR/COPII complex serves as cargo receptor for typhoid toxin.3) Although discussed in the text, Figure 6C is lacking statistical analysis. This should be included.The statistical analysis of Figure 6C was present in the figure legend of the original submission (the reviewer must have missed it).[Editors’ note: what follows is the authors’ response to the second round of review.]The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:They concur that the manuscript is significantly improved and recommended a few points to be addressed (that will not involve new experiments) prior to consideration for acceptance.Reviewer #1:In general, the authors have addressed the main concerns of the reviewers and the manuscript is much approved.As COPII is responsible for ER export there is a high possibility that blocking it through knockout could result in the mislocalisation of many proteins and subsequent off-target effects; this is not fully addressed.In addition, the authors do not discuss the extensive literature on M6PR trafficking including the role of AP1, nor do they conclusively rule out the role of AP1 in the trafficking of M6PR-bound toxin.These points could be mentioned in the ‘limitations of this study’ paragraph in the Discussion.As suggested by the reviewer we have expanded the discussion of the limitations of our studies to address the reviewer’s concern.


Lucid dreaming offers the chance to investigate dreams from within a dream and by real-time dialogue between experimenters and dreamers during REM sleep. This state of consciousness opens a new experimental venue for dream research. However, laboratory study in this field is limited due to the rarity of lucid dreamers. In a previous study, we were able to induce in 50% of the participants a lucid dream in a single sleep laboratory night by combining a wake-up-back-to-bed (WBTB) sleep routine and a mnemonic method . In three experiments, we tried to replicate our earlier findings while we adapted our procedure in shortening , simplifying , and applying another induction technique . In the three conditions, four out of 15 (26%), zero out of 20 (0%), and three out of 15 (20%) participants reported a lucid dream. Compared to the original study, the earlier sleep interruption seems to reduce the lucid dream induction rate. Furthermore, without REM awakenings in the morning, lucid dream induction failed, whereas reality testing showed a lower success rate compared to MILD. Further systematic sleep laboratory studies are needed to develop reliable techniques for lucid dream research. Lucid dreaming is defined whereby a dreaming person is fully aware of his or her current dream state and this state of consciousness often leads to increased volitional control over the ongoing dream and full access to memory . ProficiAnother promising approach seems to be the combination of a wake-up-back-to-bed sleep protocol (WBTB) and dream work . In a previous study, we conducted a sleep laboratory experiment with four distinct experimental conditions whereas the experimental design was as follows : After 6Because the aforementioned experimental procedure was very effective in inducing lucid dreams, it also lasted for up to 11 h from the beginning of the night (“lights out”) until the final awakening. Thus, we reduced the time spent in bed for the first half of the night. Instead of 6 h (4 REM periods), we reduced the time to about 4.5 h (3 REM periods) of uninterrupted sleep. This change was made for all three experiments. Furthermore, the protocol for the REM awakenings in our original study was rather complex for the second half of the nights with three different rules for REM awakenings . TherefoIn total, 73 dream reports  were collected for all experimental nights. For the first half of the night, 39 dreams  were reported after REM awakenings with a total dream recall rate of 78% . For the second half of the night, 28 dreams  were reported in the morning either after REM awakenings (Exp1 and 2) or time-based awakenings (Exp2); whereas in Experiment 1, one participant, in Experiment 2, two participants, and in Experiment 3, three participants reported two dreams. The overall dream recall rate for the number of participants is 56% . The dream reports were rated for lucidity by two independent raters. The average length of a dream report consisted of 122.9 ± 90.5 words. The interrater reliability showed almost perfect agreement in experiments 1 and 3 and substantial agreement in experiment 2, with an average weighted kappa of κ1 = 0.833, κ2 = 0.623, and κ3 = 0.925.Overall, seven participants (14%) reported lucid dreaming after WBTB. One participant (2%) was not sure whether the dream was lucid and if the LRLR-eye signal had been given. Further, 42 participants (84%) did not report a lucid dream. The judges rated 62 dream reports without evidence of lucid dreaming , four wIn experiment 1 (4.5 h + 1 h MILD + REM awakening) four out of 15 participants (26%) reported having a lucid dream. The external judges rated three as lucid dreams and one as a dream with indications of lucidity. Two of these four PSG recordings showed clear prearranged eye movement (13%), whereas the other two showed only an ambiguous (/questionable) LRLR-eye signal (13%). In experiment 2 (4.5 h + 1 h MILD + time-based awakening) none out of 20 participants reported having a lucid dream. The external judges rated none of the dream reports as lucid nor with an indication of lucidity. Hence, no eye signal could be verified. And finally in experiment 3 (4.5 h + 1 h RT + REM awakening) three out of 15 participants (20%) reported having a lucid dream. The external judges rated all three as lucid dreams. On each of these three PSG recordings, an unambiguous LRLR-eye signal could be verified (20%).The findings of the present study show that we were not able to replicate our previous results with respect to the lucid dream induction rate by applying a combination of WBTB and MILD . The lowFirstly, the sleep duration in the first half of the night was shortened from the previous 6 h to 4.5 h of sleep. The reduction of sleep duration until the WBTB intervention could lead to circadian and homeostatic differences that we did not measure in our study. In a similar sleep laboratory study with additional odor stimulation by our research group , we alsoFurthermore, due to the earlier sleep interruption, it might be the case that the participants still show high sleep pressure in the second half of the night. All participants in this study revealed high sleep efficiency (>90%) for the second half of the night, whereas in our previous study  the sleeSecondly, the induction session had been modified in the third experiment from a MILD procedure  to realiThirdly, the REM awakening procedure for the second half of the night was simplified for experiment 2 which showed no lucid dreams in a cohort of 20 participants. This null effect is rather surprising because the rest of the procedure was kept similar to all other experiments. One effect of this simplification was that the dream recall rate for the morning awakenings dropped to 20%. One might speculate that this experiment also provoked some lucid dreams. However, the participants were unable to recall the content. This assumption is supported by the general idea that a low dream recall rate results in low lucid dream rates . HoweverA final methodological limitation that needs to be addressed in future sleep lab research is the proper validation of lucid dream by LRLR eye signals, because during a night recording participants might show hundreds of eye movements during (REM) sleep. Therefore, a high probability exists to find by chance a LRLR . One metIn dream research, neural correlates are often discussed in association with dream recall rate  or withTo summarize, the present study combined the so-called wake-up-back-to-bed sleep protocol (WBTB) after 4.5 h and MILD/reality testing in three experiments to induce lucid dreams. From a total of 50 participants in three different experiments, the procedure induced in seven participants a lucid dream, whereas five of those lucid dreams were verified by an LRLR eye signal. The success rate of a combination therefore could not replicate the high success rate of other similar induction techniques in earlier studies. Future studies should focus on the methodological factors raised and their influence on lucid dream induction.In the first experiment, 15 students , in the second experiment, 20 students , and in the third experiment, 15 students  participated. The age of the total 50 participants ranged from 23 to 31 years see . All parFor obtaining data about dream and lucid dream recall frequency, the participants filled out the Mannheim Dream questionnaire (MADRE ). The drData about the sleep composition was gathered with continuous polysomnography (PSG). PSG recording included electroencephalogram , electrooculogram (EOG), submental electromyogram (EMG), including electrocardiogram (ECG). The EEG electrodes were placed according to the Ten-Twenty system . A standard sleep recording device  recorded sleep data all night. The sleep recordings were manually scored in sleep stages according to the AASM criteria . The following sleep parameters were of interest: total bed time (min), total sleep time (min), sleep efficiency (%), sleep latency (min), REM latency (min), REM period count, REM period range, REM total time (min), REM% SPT, Wake% SPT, Stage1% SPT, Stage2% SPT, Stage3% SPT.The MILD technique uses the ability of the prospect memory, which allows to set an intention or reaction based on events to be experienced in the future. This technique is the one most often tested empirically and first used in a sleep laboratory study . MILD isReality testing (RT) was first introduced by Tholey in 1982 , where hParticipants spent a single night in the sleep laboratory in Bern, in a dark and quiet room. All participants received full information regarding the procedure and aim of the studies. Written informed consent was obtained by all participants, right after they arrived in the sleep laboratory around 9:00 pm. Immediately afterwards, they were familiarized with the premises. In a next step, the electrodes for PSG were attached, and the participants prepared themselves for the night. After assuring correct sleep recording, the left-right-left-right eye movements (LRLR) were explained to the participants, followed by testing whether a clear LRLR eye signal could be successfully identified on the sleep recording, which is used to signal a possible lucid dream . Participants were informed that they would now have at least 4.5 h of uninterrupted sleep. The subsequent night procedure can be divided into four parts:First half of the night: In experiments 1 and 2, the participants were only awakened after 4.5 h of sleep and a stable REM interval of at least 10–15 min If in experiments 1 and 2 the participants did not have any stable REM of 10–15 min after 4.5 h of sleep, then they were awakened when the REM period was at least 7 min long. The limit was set after 5.5 h of falling asleep, and any REM period, no matter how long, resulted in the awakening of the participant. The last rule for awakening was that when the experimenter recognized a LRLR eye signal, then the participants were awakened three epochs (each 30 s) later.Awakening: In experiment 1, the participants were awakened by knocking on the door and calling their name until they responded. In experiments 2 and 3, the participants were called by their name via an intercom system until they responded. They were immediately asked to report any mental content that was in their mind before awakening. To assure that the participants had enough time to remember the mental content, they were given up to 2 min to recollect their dream experience. When the participant reported a dream, he was asked whether he was aware of his dream state and if he executed a LRLR eye signal. The whole conversation was recorded via voice recorder. The lights were turned on and the experimenter went in the room of the participant where the intervention was implemented.Intervention: In Experiments 1 and 2, the lucid dream induction techniques used were WBTB in combination with MILD. The participants first had to write down their dream report (15 min), which is needed for the implementation of MILD. In a second step, a motivation letter about experiencing a lucid dream in the sleep laboratory was read and filled out (5 min). The third step was to search the dream for dream clues. Therefore, the participants were given an information sheet about dream clues. Then, they had to analyze their created dream reports for such dream clues (20 min). If the participant could not remember the dream and no dream report was made, then dream reports from the dream diary were used, in which the participants had a record of their dreams for at least the three nights before the sleep laboratory night. The last step included giving the participants an information sheet about MILD. To assure that the participants had understood the meaning, they had to explain it to the experimenter. For the remaining time, MILD was practiced lying in bed (20 min), and just before turning the lights off, the LRLR eye signal was practiced again.In experiment 3, a combination of WBTB and reality testing was used to induce lucid dreaming. First, the participants were given an information sheet about lucid dreaming via reality testing (5 min). Next, the participants received information about three specific types of reality test, of which three versions existed . Versions 1–3 were equally distributed between participants. The three specific reality tests were solidified by explaining and showing them to the experimenter (25 min). Subsequently, the reality tests were repeated and internalized with two tasks. The first was watching an 11-min film sequence from “waking life” on a laptop. During that time, they had to perform, at least twice, the three reality tests in the waking world. For the remaining time, the three reality tests learned were performed in bed five times with eyes open  and five times with eyes closed (imagined dream world). The intervention in all three experiments lasted around 1 h. The time frames were used for reference only. Interindividual differences for the correct and conscientious completion of the tasks were tolerated.Second half of the night (back to bed): During the second sleep period of the participants, the experimenters of all three studies were awake. The participants were awakened three periods after the occurrence of a LRLR-eye signal, whereas the awakening followed the same procedure as in the awakening before intervention. If no LRLR-eye signal was observed by the experimenter, then different rules of awakening applied. In experiments 1 and 3 the participant was awakened after a 15-min continuous REM phase, after 3 h of falling asleep. If the REM phases were too short, then after 4 h of falling asleep, a shorter REM period resulted in the awakening of the participant. In experiment 2, the participants were awakened after every continuous REM period of 5 min. In all three studies, the dream reports were recorded with a voice recorder. In experiments 1 and 3, dream reports were also written down on a dream report sheet.The lucid dream induction counts as successful if three different types of proof hold true : (1) thThe free software “R” (version 4.0.3) was used to calculate the interrater reliability coefficient kappa to assure coherent objective rating of dream reports. Differences in mean values in the sleep data between the three groups was calculated using ANOVA  and post-hoct tests were performed with Games-Howell .