Toxoplasma gondii virulence factor ROP18 inhibits the host NF-κB pathway by promoting p65 degradation
Authors: Li Yu
Ying Chen
Qingli Luo
Yuxian Shen
Aimei Zhang
Li Cheng
Lijuan Wan
Ran An
Lijian Chen
Jilong Shen
Deyong Chu
Xiucai Xu
He Chen
Yujun Shen
Zhongru Jiang
Min Li
Jian Du
Journal: Journal of Biological Chemistry (2014)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Cell Biology; NF-kB Transcription Factor; NF-κB (NF-KB); Parasite; Parasitology
Affiliations: From the Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China.

Abstract:  The obligate intracellular parasite Toxoplasma gondii secretes effector molecules into the host cell to modulate host immunity. Previous studies have shown that T. gondii could interfere with host NF-κB signaling to promote their survival, but the effectors of type I strains remain unclear. The polymorphic rhoptry protein ROP18 is a key serine/threonine kinase that phosphorylates host proteins to modulate acute virulence. Our data demonstrated that the N-terminal portion of ROP18 is associated with the dimerization domain of p65. ROP18 phosphorylates p65 at Ser-468 and targets this protein to the ubiquitin-dependent degradation pathway. The kinase activity of ROP18 is required for p65 degradation and suppresses NF-κB activation. Consistently, compared with wild-type ROP18 strain, ROP18 kinase-deficient type I parasites displayed a severe inability to inhibit NF-κB, culminating in the enhanced production of IL-6, IL-12, and TNF-α in infected macrophages. In addition, studies have shown that transgenic parasites carrying kinase-deficient ROP18 induce M1-biased activation. These results demonstrate for the first time that the virulence factor ROP18 in T. gondii type I strains is responsible for inhibiting the host NF-κB pathway and for suppressing proinflammatory cytokine expression, thus providing a survival advantage to the infectious agent. 

Retracted: No

PubPeer comments:
 Figure 3G. * Red boxes: the left and middle GAPDH panels look similar if one of them is flipped vertically ![file] Here is a close-up comparison of both panels, with one of them flipped. The left lanes might look a bit different, but there are also remarkable similarities, in particular on the right side of the panels. ![file] , 
 Dear Elisabeth M **Bik, Thank you for your attento to our article entitled  Toxoplasma gondii virulence factor ROP18 inhibits the host NF-u03baB pathway by promoting p65 degradation, published on Journal of Biological Chemistry (2014) - 1 Comment pubmed: 24648522 doi: 10.1074 jbc.m113.544718 issn: 0021-9258 issn: 1083-351x. You mentioned that  the left and middle GAPDH panels (Fig. 3G) look similar if one of them is flipped vertically. You showed a close-up comparison of both panels, with one of them flipped, believing that there are remarkable similarities on the right side of the panels. We have checked the original figures and no mistakes were made when using the figures in manuscript processing. We also compared both papanels with care and found that a notable difference exists between both panels, even in th right half of the panel. Actually, it is impossible, for us, to change half of the blot for falcification. Please find the attached original figure (Fig 3G) for your identification. We appreciate again your comment and attention. , 
 #1 Dear Elisabeth M **Bik, Thank you for your attento to our article entitled  Toxoplasma gondii virulence factor ROP18 inhibits the host NF-u03baB pathway by promoting p65 degradation, published on Journal of Biological Chemistry (2014) - 1 Comment pubmed: 24648522 doi: 10.1074 jbc.m113.544718 issn: 0021-9258 issn: 1083-351x. You mentioned that  the left and middle GAPDH panels (Fig. 3G) look similar if one of them is flipped vertically. You showed a close-up comparison of both panels, with one of them flipped, believing that there are remarkable similarities on the right side of the panels. We have checked the original figures and no mistakes were made when using the figures in manuscript processing. We also compared both papanels with care and found that a notable difference exists between both panels, even in th right half of the panel. Actually, it is impossible, for us, to change half of the blot for falcification. Please find the attached original figure (Fig 3G) for your identification. ![file] We appreciate again your comment and attention. , 
 ![file] ![file] ![file] , 
 Der Dr. Shen: Thank you for your response to Dr. Biku2019s queries. I have still have some concerns. In figure 3G, If the two images for GAPDH are adjusted such that the bands are matched with respect to size, the spot you have labelled 1 in the ROP18-WT-GFP panel would not be included in the image in the ROP18-MUT-GFP panel; accordingly, the absence of the spot can hardly be considered a difference. While there are indeed many differences in the two images, the feature you label number 2 for example, I am struck more by features in the images that you provided that are virtually identical. I have illustrated this in what follows with arrows and circles; there are many more in addition to those that I have marked. What appear to be scratches on the film in the two images are remarkable for their identity. The two images appear to share a surprising number of background features. ![file] In addition to the similarities in the GAPDH panels noted by Dr. Bick, I am curious about the GFP panels in figure 3G (ROP18-WT-GFP and GFP vector). I show in what follows the images for ROP18-WT-GFP and GFP vector (rotated 180 degrees). While there are many obvious differences, there are some features that are remarkably similar, which I indicate in panels with red boxes. I could be convinced that these similarities are coincidental if there were higher resolution images. ![file] ![file] ![file] ![file] , 
 u662fu7684uff0cu60a8u6240u8bf4u7684uff0cu7684u786eu6709u4e00u4e9bu80ccu666fu6070u597du6bd4u8f83u76f8u4f3cu3002u5176u539fu56e0u662fuff1au6211u4eecu505aWBu7684u66ddu5149u5939u5b50u5185u90e8u51f9u51f8u4e0du5e73,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 What you mentioned that there are some backgrounds that happen to be similar. The reasons may be: due to the experiment conditions ten years ago, our WB exposure clip interior was uneven, and there were many stains (possibly the developer solution dried) in the exposure clip, resulting in different films in the same position, so contributed to a very similar background. It s just like a microscope with a scratch on the eyepiece and a picture with a similar scratch in the same place. In fact, we mainly focus on the main band, rather than such an nonspecific background. We havenot paid more attention to this previously, however, it was really done by different samples. After all, As what you said, our main bands were completely different, and our conclusions are based on the main bands, not these non-specific backgrounds. Also, Thank you! , 
 #6 Dear author, As was pointed out in comment #5, the likeliness between the first and last column of Fig. 3g (IB:GFP row) is quite striking. Please have a look at the animated overlay below. Is this similarity really expected for two independent experiments? At the very least there seems to be a rotation issue. [click to animate (181.2 degree rotation, x0.94 horizontal and x1.20 vertical)] ![file] , 
 #4 Animated comparison of the two blots from #1. Not sure what to make of it. The actual blots seem to move behind foreground lens features. The dark mist around the blots remains nearly the same. The blots are well aligned with the lens features. To obtain a match I needed to apply anisotropic scaling. This is somewhat unexpected. [ click to animate; anisotropic scaling, x: *1.08, y: *1.15 ] ![file] The middle-bottom image of Fig. 3g is in any case vertically flipped with respect to its left neighbor. In #5 it was pointed out that Fig. 3g middle-row-right panel is a 180 degree rotated version of the middle row-left panel. The authors may want to correct these issues. ![file] , 
 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 u5e76u4e14uff0cu4f60u6240u8bf4u7684u8fd9u4e24u4e2au56feu90fdu662fu5bf9u7167uff0cu672cu8eabu8fd9u4e2au5b9eu9a8cu7684u7ed3u679cu5c31u662fu4e00u4e2au9634u6027u7684u7ed3u679cuff0cu5bf9u6587u7ae0u4e3bu9898u601du60f3u6bebu65e0u5f71u54cdu3002u6211u60f3u6211u4eecu6ca1u6709u4efbu4f55u5fc5u8981u4e3au4e86u4e00u4e2au7ed3u679cu9634u6027u7684u5b9eu9a8cu7684u5bf9u7167uff0cu8d39u8fd9u4e48u5927u7684u529bu6c14u5427? u6b63u56e0u4e3au8fc7u53bbu5927u5bb6u4f7fu7528u7684u66ddu5149u5939u5b50u6709u8bf8u591au7f3au70b9uff0cu6211u4eecu5728u51e0u5e74u524du5c31u66f4u6362u6210u4e86u5168u81eau52a8u66ddu5149u4eeau4e86uff0cu4ecau540eu5728u6211u4eecu7684WBu56feu50cfu4e2duff0cu4e0du4f1au518du51fau73b0u8fd9u4e9bu975eu7279u5f02u6027u7684u80ccu666fu4e86u3002u6211u4eecu4e3au4e24u4e2au5e26u6709u975eu7279u5f02u6027u80ccu666fu7684u56feu7247u7ed9u60a8u5e26u6765u7684u56f0u60d1u611fu5230u975eu5e38u62b1u6b49u3002 Dear Orchestes Quercus, I am sorry that I didn t explain it clearly last time. As we mentioned before, due to the simple and crude experimental conditions in our lab ten years ago, the exposure clips we used before were uneven inside and had some stains, resulting in some backgrounds in the two film that happened to be similar, which make your misunderstanding. According to the images you have adjusted the exposure, upside or down, stretched or shrinked, and even animated it, anyway, we understand that you want to merge two images to each other. However, the main bands are still different. Moreover, the two images you mentioned are controls, and the result of this experiment itself is a negative result, which has no effect on the scientific idea of the study. The judgment of the experiment is based on the main band, not the non-specific background. In fact, it was not necessary to put so much endeavor into such a control in this negative experiment of such difficult study. Due to the drawback of using exposure clips, now we have changed to automatic exposure instruments recently, and we will no longer have such unspecific backgrounds on the film again. We apologize for your confusion caused by the images with similar non-specific backgrounds. We deeply appreciate your efforts for academic misconductuff01If you have any queries, please do not hesitate to contact me. Thank you! , 
 #9 Thanks for your extensive reply! You are right, the exercise in #8 is rather futile. It merely shows that the loading controls are different and that a lot of artifacts exist in the scans you made. So it shows there is no real problem. But what about comment #7? The GFP row is not a loading control, is it? The left and right column contain different images of the same gel. After 180 degree rotation, that is. , 
 Dear Orchestes Quercus, Thanks for your positive affirmations and concern. According to your suggestion, we compared the first and last column of Fig. 3g (IB: GFP row), (we only compared the main bands, because as we mentioned before, the non-specific background is caused by unevenness and staining of the exposure clips, and the results of the experiment are also judged according to the main band) and found the many differences. If you pay attention to the details and you will find they are not the rotation images, although there are some similarities. In fact, it was really done by different samples. We appreciate your efforts to the imageuff01We also apologize for your confusion caused by the images with similar non-specific backgrounds. Best regards! ![file] ![file] , 
 Dear Orchestes Quercus, Thanks for your positive affirmations and concern. According to your suggestion, we compared the first and last column of Fig. 3g (IB: GFP row), (we only compared the main bands, because as we mentioned before, the non-specific background is caused by unevenness and staining of the exposure clips, and the results of the experiment are also judged according to the main band) and found the many differences. If you pay attention to the details and you will find they are not the rotation images, although there are some similarities. In fact, it was really done by different samples. We appreciate your efforts to the imageuff01We also apologize for your confusion caused by the images with similar non-specific backgrounds. Best regards! ![file] ![file] ![file] ![file] ![file] ![file] ![file] ![file] ![file] ![file] , 
 I find the explanation given in 12 for the clear similarity of the images in figure 3G, unconvincing. I believe the differences arise because one of the two panels (perhaps both) is a composite of some kind with some elements added to make the images appear to be unrelated. The identity of the images is further disguised by rotating one of them 180 degrees. , 
 #13 I agree. I belatedly looked a bit in what one expects. GFP should appear at 27 kDa. ROP18-GFP is heavier and, according to at least the blots of these authors, apears at 90 kDa. This is exactly what Fig. 3G shows. Except that the GFP panel is clearly a rotated version of a ROP18-GFP panel. Authors, it is easy to point out differences. Can you take the time to look at and explain the obvious similarities after rotating the GFP panel by 180 degrees? Why would your dirty imaging system put that nasty stain one time at 90 kDa, 10 hr And the next time at 27 kDa, 40 hr? Perfectly aligned with the corresponding blot. Why would blots at 27 kda in ROP18-GFP suddenly move in identical pairs to 90 kDa for GFP? And can I point to the many features (stripes, dots, ...) that duplicate between the panels, see animation in #7? And please do not tell me that your system is so dirty that everything that is similar identical is perfectly blot-aligned smudge... I would be fully convinced when you would conclude that the blots are identical but imaged at e.g. a different focus and exposure time. ![file] , 
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 Dear authors, Thanks for responding. I appreciate you standing up for your work in this and other PubPeer posts. And I must be very ignorant about the procedures you use to image the NC membrane, as I really cannot see how the above two panels can be different. I understand you blame the abundant background on developer stains in, and an uneven background of, your exposure clip (cassette?). Which caused a background by emitting additional light (?). Still, if I have a dirty cassette or a bit handicapped imaging system, I would not expect a 90 kDa band in one experiment and a 27 kDa band in another experiment to align identically with the background stains. I tried to make this clear in the sketch below. Using an identical placement of the NC membrane the stains would appear at different position with respect to the bands. ![file] [ Identical ROP18-GFP 10 min and GFP 40 min stains highlighted ] ![file] [ Comparison of the 90 and 27 kDa bands wrt the stains (e.g. red dotted lines) ] ![file] In your Fig. 3G one can see that the stains match between the ROP18-GFP and GFP panels, but only after 180 degree rotation (green boxes). Moreover, the bands and individual blots are identically positioned with respect to these stains (e.g. dotted red lines). I think we can agree on this part, isn t it? You argue that you can identify differences: the details of the blots are different, some stains are bigger, others fainter .I agree with that, there are differences and this is not the same scan. Then you conclude that everything is fine because there are difference. I strongly disagree with that. There are simply too many similarities to have arisen by chance. There is no way in which positioning the NC membranes identically could lead to this result. Even rotating the GFP membrane by 180 degrees would not yield this perfect alignment of bands with stains. To me, the only valid conclusion is that we are looking at a different image of the same blot, 180 degrees rotated. How do you explain the identical alignment of the blots with the background stains for two different experiments? If you can explain it, can you possibly make some simple drawings to help me understand? More in-detail response to your points: (1) I mixed up hours and minutes. And was referencing to a specific stain by using its mass-time coordinates, (2) I did vertically scale the image to match the stains . I would argue that when the stains are caused by contamination of the cassette, they should sit at the same position. To achieve this vertical scaling stretching of the GFP panel was required. Did you vertically shrink the image? Or how do you explain the clip stains do not 1:1 match? (3) Discussed above. The position cannot have been the same as the stains would then (1) sit at a different location relative to the 90 and 27 kDa bands and (2) would not be rotated. (4) Yes, there are differences. But what about the similarities? (5) Don t understand. (6) Mostly discussed above. Although I want to add here that given the quality of the blot I have difficulty differentiating between stains and non-specific bands. This is worrying in itself. I guess that the features marked green below are bands. If you agree, I did like to know how identical non-specific bands can appear in two different experiments at different molecular weights. When you disagree, can you please mark the features that are in fact bands? ![file] , 
 According to your diagram, we found that you ve never used the exposure clip to do western-blotting. So, you can not understand what I have said. Now, I make some simple drawings to help you understand. The exposure clip is much larger than the NC membrane, so the NC membrane can be placed anywhere inside it, that is, the position of the NC membrane is not fixed in the exposure clip. This is our diagram of western-blotting, ![file] We believe that our comments that have convinced majority readers of the pubpeer. However, why can not convince you? u4eceu60a8u753bu7684u793au610fu56feu53efu4ee5u77e5u9053uff1au60a8u6ca1u6709u7528u8fc7u66ddu5149u5939u5b50u53bbu505au8fc7WB,u6240u4ee5uff0cu60a8u4e0du61c2u5f97u6211u8bf4u7684u3002 u66ddu5149u5939u5b50u662fu6bd4NCu819cu5927u5f97u591auff0cu56e0u6b64uff0cNCu819cu53efu4ee5u653eu7f6eu5728u66ddu5149u5939u5b50u5185u90e8u7684u4efbu610fu4f4du7f6euff0cu4e5fu5c31u8bf4uff0cu653eNCu819cu7684u4f4du7f6eu4e0du662fu56fau5b9au7684u3002 u8fd9u662fu6211u7684u793au610fu56feuff1a ![file] u6211u76f8u4fe1u6211u4eecu7684commentsu5df2u7ecfu80fdu591fu8bf4u670dpubpeeru7684u8bfbu8005uff0cu4f46u662fuff0cu4e0du77e5u9053u4e3au4ec0u4e48u603bu662fu4e0du80fdu8bf4u670du4f60u4eecuff1f , 
 Because in the darkroom, there is no light source, and it is also very common for the NC membrane to be placed up or down.![file] , 
 #17, #18 Thanks for your extensive reply! It is very helpful for me to understand what you have done. Ands as a spoiler, I am still not convinced. Let me explain... Let us assume that most of what you measure is stains and that only the main bands were actually present on the two NC membranes: ![file] These membranes are subsequently placed in your large and contaminated exposure clip. As you realized in #18, one of the membranes must have been rotated by 180 degrees before imaging. This then is corrected in the published image. For clarity (comparison with published images) I rotated the exposure clip instead. And just like you I find one can reproduce the two panels published panels this way: ![file] One can wonder how likely it is that the main bands of two membrane end up in a (near) identical alignment with the cover clip. So let s see what (near) identical means here. I focus on the top-right (bottom-left) blots in the ROP18-GFP (GFP) panels. The arrow points to conspicuous dots in the image: ![file] Assuming that your lanes are 10 mm wide one can get an idea of the accuracy of the co-incidental alignment by comparing the two panels (after rotation correction): [ click to animate ] ![file] The vertical red lines are 1 mm marks. Comparing the two images one can conclude that you managed to position the bands on the second membrane with a better than +-0.5 mm accuracy with respect to the stains on the cover clip. Similar results are obtained on the other side. And this without trying and without even seeing the bands. This is, with understatement, very unlikely. I can also think more highly of your WB images and assume that some of the band-shaped and lane-aligned stains actually ***are*** bands on the membrane. Assume for example that the bands shown in red below are real. This then raises the obvious question why they appear as duplicates, see the red arrow. And these are duplicates, see for example the double band at the bottom of the animated image above. ![file] So, my questions: 1. Do you think it is reasonably to believe that one accidentally positions the main bands in exactly the same position with respect to stains on a cover clip? With an accuracy of +-0.5 mm and +-0.5 degree? Without guidance and having rotated the second membrane by 180 degrees? 2. Do you really think that only the main band was present on your membranes? And that everything else you show is stains on your cover clip? Thanks for your time and explanation! , 
 I would summarize the situation as follows: - There are very few lucky people that win the lottery. - There are very few lucky people that accidentally position a pair of membranes with their bands aligned at sub-mm level. One membrane rotated ~180 degrees. And have it published. - Many more people take two images with different exposure time or magnification. Then make some [bookkeeping] [mistake] . And accidentally publish a [rotated version as something different] . ,