Disruption of mptpB impairs the ability of Mycobacterium tuberculosis to survive in guinea pigs
Authors: Anil Koul
Anil K Tyagi
V D Ramanathan
Ramandeep Singh
Aparna Khera
Amit Singh
H Shakila
Neeraj Dhar
M Naseema
C N Paramasivan
Radhika Gupta
Yogendra Singh
Vivek Rao
P R Narayanan
Journal: Molecular Microbiology (2003)
Discipline: MICROBIOLOGY
Keywords: Protein tyrosine kinases; tyrosine phosphatases; bacterial pathogens; virulence factors; phosphorylation
Affiliations: Department of Biochemistry, University of Delhi South campus, Benito Juarez Road, New Delhi-110021, India.

Abstract:  Protein tyrosine kinases and tyrosine phosphatases from several bacterial pathogens have been shown to act as virulence factors by modulating the phosphorylation and dephosphorylation of host proteins. The identification and characterization of two tyrosine phosphatases namely MptpA and MptpB from Mycobacterium tuberculosis has been reported earlier. MptpB is secreted by M. tuberculosis into extracellular mileu and exhibits a pH optimum of 5.6, similar to the pH of the lysosomal compartment of the cell. To determine the role of MptpB in the pathogenesis of M. tuberculosis, we constructed a mptpB mutant strain by homologous recombination and compared the ability of parent and the mutant strain to survive intracellularly. We show that disruption of the mptpB gene impairs the ability of the mutant strain to survive in activated macrophages and guinea pigs but not in resting macrophages suggesting the importance of its role in the host-pathogen interaction. Infection of guinea pigs with the mutant strain resulted in a 70-fold reduction in the bacillary load of spleens in infected animals as compared with the bacillary load in animals infected with the parental strain. Upon reintroduction of the mptpB gene into the mutant strain, the complemented strain was able to establish infection and survive in guinea pigs at rates comparable to the parental strain. These observations demonstrate a role of MptpB in the pathogenesis of M. tuberculosis. 

Retracted: No

PubPeer comments:
  Fig 6 7 :  Normal tissue sections for both lung and liver are identical at 3w pi (Fig. 6) a,d 6w pi (Fig. 7). The whole 6w pi panel (Fig. 7) was reused in another study as is (Singh et al., Tuberculosis, 2005). The normal lung section was reused in another study (Rao et al., 2005, Scandinavian J. Immunol.) but labeled as 8w pi. The normal liver section was used in another study (Khera et al., 2005, Vaccine) but as lung tissue. , 
  Figure 2 : bands MT1, MT2 and MT3 appear identical. Panels A and B of this figure were reused as is in another study from the same authors (Singh et al., 2005, Tuberculosis, Figs. 4A 4B). Since this figure is used to validate the mutant used in the rest of the study, its invalidity questions the whole model and conclusion of the authors. , 
  Figure 3 : Panels B and C of this figure were reused as is in another study from the same authors (Singh et al., 2005, Tuberculosis, Figs. 4C 4D). , 
 #1 **Response for statement pertaining to Fig. 6, 7 of Singh et al., 2003, Molecular Microbiology is given below:** *The normal lung tissue section shown in Fig. 6 and Fig. 7 of Singh et al., Molecular Microbiology 2003 refers to sections from uninfected unvaccinated animal. As per CPCSEA guidelines, users have the responsibility to obtain the maximum information from minimum number of animals used for experiments. Therefore, the same lung tissue from uninfected and unvaccinated animal was included in Fig. 6 and Fig. 7 just as a reference for normal tissue histology.* **Response to other parts of the above comments:** *For other parts of your comment, please see our response posted for Khera et al., 2005, Vaccine, Rao et al., 2005 Scandinavian Journal of Immunology and Singh et al., Tuberculosis 2005. * , 
 #2 **Response for the above statement - Figure 2: Bands MT1, MT2 and MT3 appear identical Is given below:** *As the first author, who has performed this experiment, I can state that the bands MT1, MT2 and MT3 in Figure 2A are not identical. For the sake of clarity to negate their presumed identical appearance, we have performed a detailed analysis of the originally submitted figure. In our analysis, the three lanes were read and viewed in MATLAB R2019a using imread and imshow programs, respectively (Figures 1A, 1C and 1E). Finally, 3-dimensional image intensity profiles were plotted for each pixel of each lane (Figures 1B, 1D and 1F respectively). In figures 1B, 1D and 1F, X-axis and Y-axis represent the corresponding pixels along X-axis and Y-axis in figures 1A, 1C and 1E, and the pixel-wise intensity is plotted along Z-axis. The analysis further confirmed that the image intensity plots for all three lanes are not identical and exhibit different patterns.*  *Further, in regard of the validity of the mutant, we have also characterized the mutant strains (MT1, MT2 and MT3) by immunoblot analysis in addition to Southern blot (These results are included in Singh et al. Molecular Microbiology, 2003). We observed no expression of MptpB in lanes corresponding to MT1, MT2 and MT3. Subsequently, MT1 was used to generate the complemented strain as evident from the restoration of expression in the complemented strain. The infection experiments in the manuscript were performed by using parental, mutant (MT1) or the complemented strain. Moreover, the MT1 strain had been validated by others in the laboratory later and was also used for the generation of multiple mutant strains. We would like to reaffirm that the characterization of the strain was performed well and the conclusions presented in the study are correct. Also, in concordance with our observations, Fan et al., have also shown that MptpB promotes M. tuberculosis survival by inhibiting the expression of inflammatory mediators and apoptosis in macrophages (PMID: 29888212). Also, MptpB inhibitors have been shown to inhibit growth of intracellular M. tuberculosis in macrophages (PMID: 19240079, 20167798) and guinea pigs (PMID: 30153005).* , 
 #3 **Response for statement pertaining to reuse of Fig. 3B and C as such in Singh et al., Tuberculosis, 2005, Fig. 4C and D.** *For the above comment, please see our response to your comment posted for Singh et al., Tuberculosis, 2005.* , 
 #5 Dear Prof. Singh, First, I d like to thank you for taking the time to read and answer my comments. In the figure you attached, JPEG compression artifacts are clearly visible on the Western blot in Figure 2A. I do not think that plotting the raw intenstity of such compressed images to underline differences is of great relevance, since there will be differences due to compression artifacts anyway, no matter how good the compression is. Between all the versions of this blot that are available, I found that the one from the Mol. Microbiol. website had the least compression, where the similarity between the bands is clearly visible (Panel A). A RGB overlay of the three bands shows a very similar shape and intensity profile with some variation (Panel B). I have performed the similar analysis (RGB profiler across 3 different y positions using ImageJ) as you did and the similary between the three bands is much more visible than on compressed images but not strictly identical, again, due to compression (Panel C). By using a different LUT for the image, general and specific similarities in background noise can also be detected, from which I have highlighted the most similar examples (again compression, not strictly identical) (Panel D).  , 
 #4  As per CPCSEA guidelines, users have the responsibility to obtain the maximum information from minimum number of animals used for experiments. I agree that animal well-being is a significant concern in basic and medical research. However, including a group of control animals in a set of experiments greatly enhances the value of results obtained from infected animals by providing a reference condition, espcially since the various results displayed in the aforementionned panel were not sampled from animals infected for the same duration. Not performing bona fide controls is unethical since animals were infected and sacrificed without a control condition to compare them to. , 
 The chief editor of Molecular Microbiology contacted us referring to the discussion on Pub Peer regarding issues related to our paper Singh et al, Mol., Microbiology 2003, including the use of same data from this paper in Singh et al., Tuberculosis, 2005. As per his suggestion, we submitted a revised Fig. 7 without lung and liver panel from uninfected unvaccinated animal as an erratum. The Chief Editor has now accepted the erratum which will be published soon. , 
 A corrigendum has been published for this paper : https: onlinelibrary.wiley.com doi 10.1111 mmi.14602  In the above article, the authors included the same images for the uninfected guinea pigs in both Figures 6 and 7. The authors request that Figure 7 be replaced by the following revised Figure 7 and revised legend. The authors emphasize that this change does not affect the conclusions reported in the paper. However, the issue of duplicated lanes raised by #2 and #7 has not been addressed by this corrigendum. COPE guidelines recommend retraction in case of clear image manipulations like this one . ,