RBMX2: A Pivotal Regulator Linking Mycobacterium bovis Infection to Epithelial-Mesenchymal Transition and Lung Cancer Progression
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eLife Assessment
The study presents a comprehensive multi-approach and functional investigation of RBMX2 as a host factor involved in Mycobacterium bovis pathogenesis and its potential role in promoting epithelial-mesenchymal transition and lung cancer progression. The findings are valuable since the possible connection between M. bovis and lung cancer and the underlying mechanisms provides a promising direction for future research. The evidence is solid with methods, data, and analyses broadly supporting the claims, albeit with minor weaknesses that, if addressed, will make the evidence stronger. The study remains of great interest to microbiology, oncology, and drug discovery scientists.
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Abstract
Abstract
The emergence and progression of tuberculosis (TB) result from the intricate interplay among the pathogen, host, and environmental factors. In 2022, there were 10.6 million new TB cases reported globally, leading to 1.3 million deaths. In regions with a high prevalence of zoonotic TB, Mycobacterium bovis (M. bovis) accounts for approximately 10% of human TB cases. The immune evasion mechanisms and latent infections of Mycobacterium tuberculosis (M. tb) complicate our understanding of the host immune response to TB. This study identifies a novel host factor, RNA-binding motif protein X-linked 2 (RBMX2), which shows potential against M. bovis infection. However, the specific molecular mechanisms and roles of RBMX2 during M. bovis infection remain poorly understood. Our investigations revealed that following infection, RBMX2 was highly expressed in various cell types, including embryonic bovine lung (EBL) cells, bovine macrophage (BoMac) cells, bovine lung alveolar primary cells, and human pulmonary alveolar epithelial cells (A549). Using a multifaceted approach that included global transcriptional sequencing, proteomics, cell adhesion assays, ChIP-PCR, and Western blot analyses, we demonstrated that RBMX2 inhibits cell adhesion and tight junction formation in EBL cells while promoting the adhesion and invasion of M. bovis through the activation of the p65 pathway. Furthermore, our data suggest that RBMX2 regulates epithelial-mesenchymal transition (EMT), a process strongly associated with cancer, as indicated by our global transcriptomics, proteomics, and metabolomics analyses. To further explore the relationship between RBMX2 and cancer, we analyzed the TIMER2.0 database and found elevated expression levels of RBMX2 in lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) tissues compared to normal lung tissues. This finding was corroborated by immunofluorescence validation. After constructing an M. bovis-infected BoMac-induced EBL-EMT model, we confirmed that RBMX2 contributes to EMT by activating the p65/MMP-9 pathway post-infection.
This study elucidates the role of RBMX2 as a novel host factor with potential anti-TB functions that inhibit TB-induced EMT. These insights have vital implications for the development of TB vaccines and therapeutic strategies for TB-mediated lung cancer, highlighting RBMX2 as a promising target for future research.
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eLife Assessment
The study presents a comprehensive multi-approach and functional investigation of RBMX2 as a host factor involved in Mycobacterium bovis pathogenesis and its potential role in promoting epithelial-mesenchymal transition and lung cancer progression. The findings are valuable since the possible connection between M. bovis and lung cancer and the underlying mechanisms provides a promising direction for future research. The evidence is solid with methods, data, and analyses broadly supporting the claims, albeit with minor weaknesses that, if addressed, will make the evidence stronger. The study remains of great interest to microbiology, oncology, and drug discovery scientists.
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Reviewer #1 (Public review):
Summary:
This manuscript presents a compelling study identifying RBMX2 as a novel host factor upregulated during Mycobacterium bovis infection.
The study demonstrates that RBMX2 plays a role in:
(1) Facilitating M. bovis adhesion, invasion, and survival in epithelial cells.
(2) Disrupting tight junctions and promoting EMT.
(3) Contributing to inflammatory responses and possibly predisposing infected tissue to lung cancer development.
By using a combination of CRISPR-Cas9 library screening, multi-omics, coculture models, and bioinformatics, the authors establish a detailed mechanistic link between M. bovis infection and cancer-related EMT through the p65/MMP-9 signaling axis. Identification of RBMX2 as a bridge between TB infection and EMT is novel.
Strengths:
This topic and data are both novel and …
Reviewer #1 (Public review):
Summary:
This manuscript presents a compelling study identifying RBMX2 as a novel host factor upregulated during Mycobacterium bovis infection.
The study demonstrates that RBMX2 plays a role in:
(1) Facilitating M. bovis adhesion, invasion, and survival in epithelial cells.
(2) Disrupting tight junctions and promoting EMT.
(3) Contributing to inflammatory responses and possibly predisposing infected tissue to lung cancer development.
By using a combination of CRISPR-Cas9 library screening, multi-omics, coculture models, and bioinformatics, the authors establish a detailed mechanistic link between M. bovis infection and cancer-related EMT through the p65/MMP-9 signaling axis. Identification of RBMX2 as a bridge between TB infection and EMT is novel.
Strengths:
This topic and data are both novel and significant, expanding the understanding of transcriptomic diversity beyond RBM2 in M. bovis responsive functions.
Weaknesses:
(1) The abstract and introduction sometimes suggest RBMX2 has protective anti-TB functions, yet results show it facilitates pathogen adhesion and survival. The authors need to rephrase claims to avoid contradiction.
(2) While p65/MMP-9 is convincingly implicated, the role of MAPK/p38 and JNK is less clearly resolved.
(3) Metabolomics results are interesting but not integrated deeply into the main EMT narrative.
(4) A key finding and starting point of this study is the upregulation of RBMX2 upon M. bovis infection. However, the authors have only assessed RBMX2 expression at the mRNA level following infection with M. bovis and BCG. To strengthen this conclusion, it is essential to validate RBMX2 expression at the protein level through techniques such as Western blotting or immunofluorescence. This would significantly enhance the credibility and impact of the study's foundational observation.
(5) The manuscript would benefit from a more in-depth discussion of the relationship between tuberculosis (TB) and lung cancer. While the study provides experimental evidence suggesting a link via EMT induction, integrating current literature on the epidemiological and mechanistic connections between chronic TB infection and lung tumorigenesis would provide important context and reinforce the translational relevance of the findings.
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Reviewer #2 (Public review):
Summary:
I am not familiar with cancer biology, so my review mainly focuses on the infection part of the manuscript. Wang et al identified an RNA-binding protein RBMX2 that links the Mycobacterium bovis infection to the epithelial-Mesenchymal transition and lung cancer progression. Upon mycobacterium infection, the expression of RBMX2 was moderately increased in multiple bovine and human cell lines, as well as bovine lung and liver tissues. Using global approaches, including RNA-seq and proteomics, the authors identified differential gene expression caused by the RBMX2 knockout during M. bovis infection. Knockout of RBMX2 led to significant upregulations of tight-junction related genes such as CLDN-5, OCLN, ZO-1, whereas M. bovis infection affects the integrity of epithelial cell tight junctions and …
Reviewer #2 (Public review):
Summary:
I am not familiar with cancer biology, so my review mainly focuses on the infection part of the manuscript. Wang et al identified an RNA-binding protein RBMX2 that links the Mycobacterium bovis infection to the epithelial-Mesenchymal transition and lung cancer progression. Upon mycobacterium infection, the expression of RBMX2 was moderately increased in multiple bovine and human cell lines, as well as bovine lung and liver tissues. Using global approaches, including RNA-seq and proteomics, the authors identified differential gene expression caused by the RBMX2 knockout during M. bovis infection. Knockout of RBMX2 led to significant upregulations of tight-junction related genes such as CLDN-5, OCLN, ZO-1, whereas M. bovis infection affects the integrity of epithelial cell tight junctions and inflammatory responses. This study establishes that RBMX2 is an important host factor that modulates the infection process of M. bovis.
Strengths:
(1) This study tested multiple types of bovine and human cells, including macrophages, epithelial cells, and clinical tissues at multiple timepoints, and firmly confirmed the induced expression of RBMX2 upon M. bovis infection.
(2) The authors have generated the monoclonal RBMX2 knockout cell lines and comprehensively characterized the RBMX2-dependent gene expression changes using a combination of global omics approaches. The study has validated the impact of RBMX2 knockout on the tight-junction pathway and on the M. bovis infection, establishing RBMX2 as a crucial host factor.
Weaknesses:
(1) The RBMX2 was only moderately induced (less than 2-fold) upon M. bovis infection, arguing its contribution may be small. Its value as a therapeutic target is not justified. How RBMX2 was activated by M. bovis infection was unclear.
(2) Although multiple time points have been included in the study, most analyses lack temporal resolution. It is difficult to appreciate the impact/consequence of M. bovis infection on the analyzed pathways and processes.
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Reviewer #3 (Public review):
Summary:
This study investigates the role of the host protein RBMX2 in regulating the response to Mycobacterium bovis infection and its connection to epithelial-mesenchymal transition (EMT), a key pathway in cancer progression. Using bovine and human cell models, the authors have wisely shown that RBMX2 expression is upregulated following M. bovis infection and promotes bacterial adhesion, invasion, and survival by disrupting epithelial tight junctions via the p65/MMP-9 signaling pathway. They also demonstrate that RBMX2 facilitates EMT and is overexpressed in human lung cancers, suggesting a potential link between chronic infection and tumor progression. The study highlights RBMX2 as a novel host factor that could serve as a therapeutic target for both TB pathogenesis and infection-related cancer risk.
Stren…
Reviewer #3 (Public review):
Summary:
This study investigates the role of the host protein RBMX2 in regulating the response to Mycobacterium bovis infection and its connection to epithelial-mesenchymal transition (EMT), a key pathway in cancer progression. Using bovine and human cell models, the authors have wisely shown that RBMX2 expression is upregulated following M. bovis infection and promotes bacterial adhesion, invasion, and survival by disrupting epithelial tight junctions via the p65/MMP-9 signaling pathway. They also demonstrate that RBMX2 facilitates EMT and is overexpressed in human lung cancers, suggesting a potential link between chronic infection and tumor progression. The study highlights RBMX2 as a novel host factor that could serve as a therapeutic target for both TB pathogenesis and infection-related cancer risk.
Strengths:
The major strengths lie in its multi-omics integration (transcriptomics, proteomics, metabolomics) to map RBMX2's impact on host pathways, combined with rigorous functional assays (knockout/knockdown, adhesion/invasion, barrier tests) that establish causality through the p65/MMP-9 axis. Validation across bovine and human cell models and in clinical tissue samples enhances translational relevance. Finally, identifying RBMX2 as a novel regulator linking mycobacterial infection to EMT and cancer progression opens exciting therapeutic avenues.
Weaknesses:
Although it's a solid study, there are a few weaknesses noted below.
(1) In the transcriptomics analysis, the authors performed (GO/KEGG) to explore biological functions. Did they perform the search locally or globally? If the search was performed with a global reference, then I would recommend doing a local search. That would give more relevant results. What is the logic behind highlighting some of the enriched pathways (in red), and how are they relevant to the current study?
(2) While the authors show that RBMX2 expression correlates with EMT-related gene expression and barrier dysfunction, the evidence for direct association remains limited in this study. How does RBMX2 activate p65? Does it bind directly to p65 or modulate any upstream kinases? Could ChIP-seq or CLIP-seq provide further evidence for direct RNA or DNA targets of RBMX2 that drive EMT or NF-κB signaling?
(3) The manuscript suggests that RBMX2 enhances adhesion/invasion of several bacterial species (e.g., E. coli, Salmonella), not just M. bovis. This raises questions about the specificity of RBMX2's role in Mycobacterium-specific pathogenesis. Is RBMX2 a general epithelial barrier regulator or does it exhibit preferential effects in mycobacterial infection contexts? How does this generality affect its potential as a TB-specific therapeutic target?
(4) The quality of the figures is very poor. High-resolution images should be provided.
(5) The methods are not very descriptive, particularly the omics section.
(6) The manuscript is too dense, with extensive multi-omics data (transcriptomics, proteomics, metabolomics) but relatively little mechanistic integration. The authors should have focused on the key mechanistic pathways in the figures. Improving the narratives in the Results and Discussion section could help readers follow the logic of the experimental design and conclusions.
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Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
This manuscript presents a compelling study identifying RBMX2 as a novel host factor upregulated during Mycobacterium bovis infection.
The study demonstrates that RBMX2 plays a role in:
(1) Facilitating M. bovis adhesion, invasion, and survival in epithelial cells.
(2) Disrupting tight junctions and promoting EMT.
(3) Contributing to inflammatory responses and possibly predisposing infected tissue to lung cancer development.
By using a combination of CRISPR-Cas9 library screening, multi-omics, coculture models, and bioinformatics, the authors establish a detailed mechanistic link between M. bovis infection and cancer-related EMT through the p65/MMP-9 signaling axis. Identification of RBMX2 as a bridge between TB infection and EMT is novel.
Strengths:
Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
This manuscript presents a compelling study identifying RBMX2 as a novel host factor upregulated during Mycobacterium bovis infection.
The study demonstrates that RBMX2 plays a role in:
(1) Facilitating M. bovis adhesion, invasion, and survival in epithelial cells.
(2) Disrupting tight junctions and promoting EMT.
(3) Contributing to inflammatory responses and possibly predisposing infected tissue to lung cancer development.
By using a combination of CRISPR-Cas9 library screening, multi-omics, coculture models, and bioinformatics, the authors establish a detailed mechanistic link between M. bovis infection and cancer-related EMT through the p65/MMP-9 signaling axis. Identification of RBMX2 as a bridge between TB infection and EMT is novel.
Strengths:
This topic and data are both novel and significant, expanding the understanding of transcriptomic diversity beyond RBM2 in M. bovis responsive functions.
Weaknesses:
(1) The abstract and introduction sometimes suggest RBMX2 has protective anti-TB functions, yet results show it facilitates pathogen adhesion and survival. The authors need to rephrase claims to avoid contradiction.
We sincerely appreciate the reviewer's valuable feedback regarding the need to clarify RBMX2's role throughout the manuscript. We have carefully revised the text to ensure consistent messaging about RBMX2's function in promoting M. bovis infection. Below we detail the specific modifications made:
(1) Introduction Revisions:
Changed "The objective of this study was to elucidate the correlation between host genes and the susceptibility of M.bovis infection" to "The objective of this study was to identify host factors that promote susceptibility to M.bovis infection"
Revised "RBMX2 polyclonal and monoclonal cell lines exhibited favorable phenotypes" to "RBMX2 knockout cell lines showed reduced bacterial survival"
Replaced "The immune regulatory mechanism of RBMX2" with "The role of RBMX2 in facilitating M.bovis immune evasion"
(2) Results Revisions:
Modified "RBMX2 fails to affect cell morphology and the ability to proliferate and promotes M.bovis infection" to "RBMX2 does not alter cell viability but significantly enhances M.bovis infection"
Strengthened conclusion in Figure 4: "RBMX2 actively disrupts tight junctions to facilitate bacterial invasion"
(3) Discussion Revisions:
Revised screening description: "We screened host factors affecting M.bovis susceptibility and identified RBMX2 as a key promoter of infection"
Strengthened concluding statement: "In summary, RBMX2 drives TB pathogenesis by compromising epithelial barriers and inducing EMT"
These targeted revisions ensure that:
All sections consistently present RBMX2 as promoting infection; the language aligns with our experimental finding; potential protective interpretations have been eliminated. We believe these modifications have successfully addressed the reviewer's concern while maintaining the manuscript's original structure and scientific content. We appreciate the opportunity to improve our manuscript and thank the reviewer for this constructive suggestion.
(2) >While p65/MMP-9 is convincingly implicated, the role of MAPK/p38 and JNK is less clearly resolved.
We sincerely appreciate the reviewer's insightful comment regarding the roles of MAPK/p38 and JNK in our study. Our experimental data clearly demonstrated that RBMX2 knockout significantly reduced phosphorylation levels of p65, p38, and JNK (Fig. 5A), indicating potential involvement of all three pathways in RBMX2-mediated regulation.
Through systematic functional validation, we obtained several important findings:
In pathway inhibition experiments, p65 activation (PMA treatment) showed the most dramatic effects on both tight junction disruption (ZO-1, OCLN reduction) and EMT marker regulation (E-cadherin downregulation, N-cadherin upregulation);
p38 activation (ML141 treatment) exhibited moderate effects on these processes;
JNK activation (Anisomycin treatment) displayed minimal impact.
Most conclusively, siRNA-mediated silencing of p65 alone was sufficient to:
Restore epithelial barrier function
Reverse EMT marker expression
Reduce bacterial adhesion and invasion
These results establish a clear hierarchy in pathway importance: p65 serves as the primary mediator of RBMX2's effects, while p38 plays a secondary role and JNK appears non-essential under our experimental conditions. We have now clarified this relationship in the revised Discussion section to strengthen this conclusion.
This refined understanding of pathway hierarchy provides important mechanistic insights while maintaining consistency with all our experimental data. We thank the reviewer for this valuable suggestion that helped improve our manuscript.
(3) Metabolomics results are interesting but not integrated deeply into the main EMT narrative.
Thank you for this constructive suggestion. In this article, we detected the metabolome of RBMX2 knockout and wild-type cells after Mycobacterium bovis infection, which mainly served as supporting evidence for our EMT model. However, we did not conduct an in-depth discussion of these findings. We have now added a detailed discussion of this section to further support our EMT model.
ADD:Meanwhile, metabolic pathways enriched after RBMX2 deletion, such as nucleotide metabolism, nucleotide sugar synthesis, and pentose interconversion, primarily support cell proliferation and migration during EMT by providing energy precursors, regulating glycosylation modifications, and maintaining redox balance; cofactor synthesis and amino sugar metabolism participate in EMT regulation through influencing metabolic remodeling and extracellular matrix interactions; chemokine and cGMP-PKG signaling pathways may further mediate inflammatory responses and cytoskeletal rearrangements, collectively promoting the EMT process.
(4) A key finding and starting point of this study is the upregulation of RBMX2 upon M. bovis infection. However, the authors have only assessed RBMX2 expression at the mRNA level following infection with M. bovis and BCG. To strengthen this conclusion, it is essential to validate RBMX2 expression at the protein level through techniques such as Western blotting or immunofluorescence. This would significantly enhance the credibility and impact of the study's foundational observation.
Thank you for your comment. We have supplemented the experiments in this part and found that Mycobacterium bovis infection can significantly enhance the expression level of RBMX2 protein.
(5) The manuscript would benefit from a more in-depth discussion of the relationship between tuberculosis (TB) and lung cancer. While the study provides experimental evidence suggesting a link via EMT induction, integrating current literature on the epidemiological and mechanistic connections between chronic TB infection and lung tumorigenesis would provide important context and reinforce the translational relevance of the findings.
We sincerely appreciate the valuable comments from the reviewer. We fully agree with your suggestion to further explore the relationship between tuberculosis (TB) and lung cancer. In the revised manuscript, we will add a new paragraph in the Discussion section to systematically integrate the current literature on the epidemiological and mechanistic links between chronic tuberculosis infection and lung cancer development, including the potential bridging roles of chronic inflammation, tissue damage repair, immune microenvironment remodeling, and the epithelial-mesenchymal transition (EMT) pathway. This addition will help more comprehensively interpret the clinical implications of the observed EMT activation in the context of our study, thereby enhancing the biological plausibility and clinical translational value of our findings.
ADD:There is growing epidemiological evidence suggesting that chronic TB infection represents a potential risk factor for the development of lung cancer. Studies have shown that individuals with a history of TB exhibit a significantly increased risk of lung cancer, particularly in areas of the lung with pre-existing fibrotic scars, indicating that chronic inflammation, tissue repair, and immune microenvironment remodeling may collectively contribute to malignant transformation 74. Moreover, EMT not only endows epithelial cells with mesenchymal features that enhance migratory and invasive capacity but is also associated with the acquisition of cancer stem cell-like properties and therapeutic resistance 75. Therefore, EMT may serve as a crucial molecular link connecting chronic TB infection with the malignant transformation of lung epithelial cells, warranting further investigation in the intersection of infection and tumorigenesis.
Reviewer #2 (Public review):
Summary:
I am not familiar with cancer biology, so my review mainly focuses on the infection part of the manuscript. Wang et al identified an RNA-binding protein RBMX2 that links the Mycobacterium bovis infection to the epithelial-Mesenchymal transition and lung cancer progression. Upon mycobacterium infection, the expression of RBMX2 was moderately increased in multiple bovine and human cell lines, as well as bovine lung and liver tissues. Using global approaches, including RNA-seq and proteomics, the authors identified differential gene expression caused by the RBMX2 knockout during M. bovis infection. Knockout of RBMX2 led to significant upregulations of tight-junction related genes such as CLDN-5, OCLN, ZO-1, whereas M. bovis infection affects the integrity of epithelial cell tight junctions and inflammatory responses. This study establishes that RBMX2 is an important host factor that modulates the infection process of M. bovis.
Strengths:
(1) This study tested multiple types of bovine and human cells, including macrophages, epithelial cells, and clinical tissues at multiple timepoints, and firmly confirmed the induced expression of RBMX2 upon M. bovis infection.
(2) The authors have generated the monoclonal RBMX2 knockout cell lines and comprehensively characterized the RBMX2-dependent gene expression changes using a combination of global omics approaches. The study has validated the impact of RBMX2 knockout on the tight-junction pathway and on the M. bovis infection, establishing RBMX2 as a crucial host factor.
Weaknesses:
(1) The RBMX2 was only moderately induced (less than 2-fold) upon M. bovis infection, arguing its contribution may be small. Its value as a therapeutic target is not justified. How RBMX2 was activated by M. bovis infection was unclear.
Thank you for your valuable and constructive comments. In this study, we primarily utilized the CRISPR whole-genome screening approach to identify key factors involved in bovine tuberculosis infection. Through four rounds of screening using a whole-genome knockout cell line of bovine lung epithelial cells infected with Mycobacterium bovis, we identified RBMX2 as a critical factor.
Although the transcriptional level change of RBMX2 was less than two-fold, following the suggestion of Reviewer 1, we examined its expression at the protein level, where the change was more pronounced, and we have added these results to the manuscript.
Regarding the mechanism by which RBMX2 is activated upon M. bovis infection, we previously screened for interacting proteins using a Mycobacterium tuberculosis secreted and membrane protein library, but unfortunately, we did not identify any direct interacting proteins from M. tuberculosis (https://doi.org/10.1093/nar/gkx1173).
(2) Although multiple time points have been included in the study, most analyses lack temporal resolution. It is difficult to appreciate the impact/consequence of M. bovis infection on the analyzed pathways and processes.
We appreciate the valuable comments from the reviewers. Although our study included multiple time points post-infection, in our experimental design we focused on different biological processes and phenotypes at distinct time points:
During the early phase (e.g., 2 hours post-infection), we focused on barrier phenotypes; during the intermediate phase (e.g., 24 hours post-infection), we concentrated more on pathway activation and EMT phenotypes;
And during the later phase (e.g., 48–72 hours post-infection), we focused more on cell death phenotypes, which were validated in another FII article (https://doi.org/10.3389/fimmu.2024.1431207).
We also examined the impact of varying infection durations on RBMX2 knockout EBL cellular lines via GO analysis. At 0 hpi, genes were primarily related to the pathways of cell junctions, extracellular regions, and cell junction organization. At 24 hpi, genes were mainly associated with pathways of the basement membrane, cell adhesion, integrin binding and cell migration By 48 hpi, genes were annotated into epithelial cell differentiation and were negatively regulated during epithelial cell proliferation. This indicated that RBMX2 can regulate cellular connectivity throughout the stages of M. bovis infection.
For KEGG analysis, genes linked to the MAPK signaling pathway, chemical carcinogen-DNA adducts, and chemical carcinogen-receptor activation were observed at 0 hpi. At 24 hpi, significant enrichment was found in the ECM-receptor interaction, PI3K-Akt signaling pathway, and focal adhesion. Upon enrichment analysis at 48 hpi, significant enrichment was noted in the TGF-beta signaling pathway, transcriptional misregulation in cancer, microRNAs in cancer, small cell lung cancer, and p53 signaling pathway.
Reviewer #3 (Public review):
Summary:
This study investigates the role of the host protein RBMX2 in regulating the response to Mycobacterium bovis infection and its connection to epithelial-mesenchymal transition (EMT), a key pathway in cancer progression. Using bovine and human cell models, the authors have wisely shown that RBMX2 expression is upregulated following M. bovis infection and promotes bacterial adhesion, invasion, and survival by disrupting epithelial tight junctions via the p65/MMP-9 signaling pathway. They also demonstrate that RBMX2 facilitates EMT and is overexpressed in human lung cancers, suggesting a potential link between chronic infection and tumor progression. The study highlights RBMX2 as a novel host factor that could serve as a therapeutic target for both TB pathogenesis and infection-related cancer risk.
Strengths:
The major strengths lie in its multi-omics integration (transcriptomics, proteomics, metabolomics) to map RBMX2's impact on host pathways, combined with rigorous functional assays (knockout/knockdown, adhesion/invasion, barrier tests) that establish causality through the p65/MMP-9 axis. Validation across bovine and human cell models and in clinical tissue samples enhances translational relevance. Finally, identifying RBMX2 as a novel regulator linking mycobacterial infection to EMT and cancer progression opens exciting therapeutic avenues.
Weaknesses:
Although it's a solid study, there are a few weaknesses noted below.
(1) In the transcriptomics analysis, the authors performed (GO/KEGG) to explore biological functions. Did they perform the search locally or globally? If the search was performed with a global reference, then I would recommend doing a local search. That would give more relevant results. What is the logic behind highlighting some of the enriched pathways (in red), and how are they relevant to the current study?
We appreciate the reviewer's thoughtful questions regarding our transcriptomic analysis. In this study, we employed a localized enrichment approach focusing specifically on gene expression profiles from our bovine lung epithelial cell system. This cell-type-specific analysis provides more biologically relevant results than global database searches alone.
Regarding the highlighted pathways, these represent:
(1) Temporally significant pathways showing strongest enrichment at each stage:
0h: Cell junction organization (immediate barrier response)
24h: ECM-receptor interaction (early EMT initiation)
48h: TGF-β signaling (chronic remodeling)
(2) Mechanistically linked to our core findings about RBMX2's role in:
Epithelial barrier disruption
Mesenchymal transition
Chronic infection outcomes
We selected these particular pathways because they:
(1) Showed the most statistically significant changes (FDR <0.001)
(2) Formed a coherent biological narrative across infection stages
(3) Were independently validated in our functional assays
This targeted approach allows us to focus on the most infection-relevant pathways while maintaining statistical rigor.
(2) While the authors show that RBMX2 expression correlates with EMT-related gene expression and barrier dysfunction, the evidence for direct association remains limited in this study. How does RBMX2 activate p65? Does it bind directly to p65 or modulate any upstream kinases? Could ChIP-seq or CLIP-seq provide further evidence for direct RNA or DNA targets of RBMX2 that drive EMT or NF-κB signaling?
We sincerely appreciate the reviewer's in-depth questions regarding the mechanisms by which RBMX2 activates p65 and its association with EMT. Although the molecular mechanism remains to be fully elucidated, our study has provided experimental evidence supporting a direct regulatory relationship between RBMX2 and the p65 subunit of the NF-κB pathway. Specifically, we investigated whether the transcription factor p65 could directly bind to the promoter region of RBMX2 using CHIP experiments. The results demonstrated that the transcription factor p65 can physically bind to the RBMX2 region.
Furthermore, dual-luciferase reporter assays were conducted, showing that p65 significantly enhances the transcriptional activity of the RBMX2 promoter, indicating a direct regulatory effect of RBMX2 on p65 expression.
These findings support our hypothesis that RBMX2 activates the NF-κB signaling pathway through direct interaction with the p65 protein, thereby participating in the regulation of EMT progression and barrier function.
In our subsequent work papers, we will also employ experiments such as CLIP to further investigate the specific mechanisms through which RBMX2 exerts its regulatory functions.
(3) The manuscript suggests that RBMX2 enhances adhesion/invasion of several bacterial species (e.g., E. coli, Salmonella), not just M. bovis. This raises questions about the specificity of RBMX2's role in Mycobacterium-specific pathogenesis. Is RBMX2 a general epithelial barrier regulator or does it exhibit preferential effects in mycobacterial infection contexts? How does this generality affect its potential as a TB-specific therapeutic target?
Thank you for your valuable comments. When we initially designed this experiment, we were interested in whether the RBMX2 knockout cell line could confer effective resistance not only against Mycobacterium bovis but also against Gram-negative and Gram-positive bacteria. Surprisingly, we indeed observed resistance to the invasion of these pathogens, albeit weaker compared to that against Mycobacterium bovis.
Nevertheless, we believe these findings merit publication in eLife. Moreover, RBMX2 knockout does not affect the phenotype of epithelial barrier disruption under normal conditions; its significant regulatory effect on barrier function is only evident upon infection with Mycobacterium bovis.
Importantly, during our genome-wide knockout library screening, RBMX2 was not identified in the screening models for Salmonella or Escherichia coli, but was consistently detected across multiple rounds of screening in the Mycobacterium bovis model.
(4) The quality of the figures is very poor. High-resolution images should be provided.
Thank you for your feedback; we provided higher-resolution images.
(5) The methods are not very descriptive, particularly the omics section.
Thank you for your comments; we have revised the description of the sequencing section.
(6) The manuscript is too dense, with extensive multi-omics data (transcriptomics, proteomics, metabolomics) but relatively little mechanistic integration. The authors should have focused on the key mechanistic pathways in the figures. Improving the narratives in the Results and Discussion section could help readers follow the logic of the experimental design and conclusions.
Thank you for your valuable comments. We have streamlined the figures and revised the description of the results section accordingly.
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