The resurgence of M. bovis-associated TB presents a substantial global challenge, affecting both livestock and human populations. Notably, M. bovis exhibits over 99% nucleotide similarity with M. tb (Reis and Cunha, 2021). The pathogenesis of TB is a complex process influenced by a confluence of bacterial, host, and environmental factors. Both M. tb and M. bovis have evolved sophisticated strategies to evade host immune responses, enabling their long-term intracellular persistence. It is estimated that approximately one-third of the global population harbors latent TB infections (Fan et al., 2018; Borkowska et al., 2017).
The successful establishment of infection by mycobacteria is contingent upon their ability to circumvent early innate immune defenses, employing both transcriptional and post-transcriptional regulatory strategies within host macrophages (Rastogi et al., 2023; Corleis and Dorhoi, 2020; Abebe, 2021; Reuschl et al., 2017; Dorhoi and Kaufmann, 2014). Despite notable advancements in the field, unraveling the mechanisms of immune evasion and understanding the dynamics of latent infections caused by M. tb and M. bovis remains a substantial challenge. The cellular immune response to these pathogens is inherently complex, further compounded by the microdiversity of mycobacteria within individual hosts and the variability of immune responses among different individuals (Abebe, 2021; Rajaram et al., 2014).
Lung epithelial cells serve as the primary physical barrier against infection and play a pivotal role in the innate immune response. These cells not only function as a frontline defense but also recruit and activate antigen-presenting cells, such as macrophages, to initiate adaptive immune responses against M. bovis infection (D’Agnillo et al., 2021; Barros et al., 2022). Given that alveolar epithelial cells can act as reservoirs for M. bovis, their role in the infection process is particularly significant. Additionally, activated macrophages and neutrophils enhance the bactericidal effects of alveolar epithelial cells, further contributing to the host’s defense mechanisms (Hu et al., 2023; Buckley and Turner, 2018).
In our study, we employed a CRISPR–Cas9 mutant library generated in our laboratory to explore potential host factors involved in M. bovis infection. Notably, we identified RBMX2 as a protein that significantly promotes M. bovis infection. The RBMX2 protein, characterized by an RNA recognition motif within its 56–134 amino acid residue region, is implicated in mRNA splicing through spliceosomes and is emerging as a potential molecular marker for sperm activity (Ahmadi Rastegar et al., 2015). The downregulation of RBMX2 in the X chromosome of lung telocytes suggests its involvement in cellular immunity (Zhu et al., 2015).
Further exploration of RBM genes in cattle revealed that EIF3G, RBM14, RBM42, RBMX44, RBM17, PUF60, SART3, and RBM25 belong to the same subfamily as RBMX2. Previous studies have demonstrated that genes within this subfamily can regulate the proliferation and lifecycle of cancer cells. For instance, EIF3G modulates the mTOR signaling pathway, inhibiting the proliferation and metastasis of bladder cancer cells (Zhang et al., 2021). Similarly, RBM14 has been linked to the reprogramming of glycolysis in lung cancer, acting as a novel epigenetically activated oncogene (Hu et al., 2023). Despite these insights, the specific function of RBMX2 in the context of M. bovis infection and its potential role in cancer pathogenesis remains largely unexplored.
Our findings reveal that RBMX2 is upregulated in TB-infected cells, demonstrating its capacity to enhance M. bovis infection. Transcriptomic analyses suggest that RBMX2 may disrupt tight junctions within epithelial cells and promote EMT following M. bovis infection.
Epithelial cells serve as more than passive barriers; they actively participate in innate immunity by regulating cytokine secretion and maintaining barrier integrity (Buckley and Turner, 2018). Disruptions in epithelial tight junctions, often exacerbated by pro-inflammatory stimuli from pathogenic bacteria, can facilitate bacterial translocation and subsequent infection (Buckley and Turner, 2018; Savagner, 2015; Kyuno et al., 2021).
In our experiments, we observed that the disruption of the epithelial barrier facilitated M. bovis adhesion and invasion. Conversely, the knockout of RBMX2 stabilized the epithelial barrier, attenuating M. bovis invasion and intracellular survival. This stabilization also mitigated downstream innate immune responses, reducing cellular inflammation, ROS production, and apoptosis.
The loss of tight junction integrity is a precursor to EMT, a process increasingly recognized for its role in cancer progression (Li et al., 2021; Savagner, 2015; Kyuno et al., 2021; Hashimoto and Oshima, 2022). Recent studies have established a link between bacterial infections and EMT induction, particularly in the context of gastric adenocarcinomas (Malfertheiner et al., 2023; Brito et al., 2019). Epidemiological evidence suggests that TB may serve as a risk factor for lung cancer (Ho and Leung, 2018; Christopoulos et al., 2014); however, the underlying cellular mechanisms remain elusive. Chronic TB infection has been implicated in lung carcinogenesis, with reports indicating that BCG vaccination can enhance the survival of tumor cells under inflammatory conditions (Nalbandian et al., 2009; Holla et al., 2014). 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 (Hwang et al., 2022). 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 (Brabletz, 2012). 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.
Moreover, M. tb-infected THP-1 cells have been shown to induce EMT in LUAD epithelial cells (Gupta et al., 2016). Chronic infection with M. tb is associated with oxidative stress and inflammatory cytokine production, fostering an environment conducive to tumor progression (Leung et al., 2020; de la Barrera et al., 2004). Our analysis of RBMX2 across various cancers revealed increased expression levels in LUAD and LUSC, suggesting a conserved role in tumor biology across species.
In light of these findings, we constructed a model of M. bovis infection in EBL cells to investigate EMT induction. Initial results indicated that M. bovis alone did not induce EMT; however, a coculture model incorporating M. bovis-infected BoMac cells successfully induced EMT in EBL cells. Notably, the knockout of RBMX2 in this context inhibited EMT, suggesting that RBMX2 may elevate the risk of lung cancer through EMT induction following M. bovis infection. Meanwhile, metabolic pathways enriched after RBMX2 knockout, 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.
In summary, RBMX2 drives TB pathogenesis by compromising epithelial barriers and inducing EMT. Targeting RBMX2 may present a promising avenue for the prevention and treatment of TB in both humans and animals. Additionally, the effective modulation of RBMX2 could potentially mitigate the incidence of TB-associated EMT and its implications for lung cancer development in the near future.
Conclusion
Our research findings indicate that RBMX2 significantly enhances the invasive capacity of M. bovis by promoting the activation and nuclear translocation of the p65 protein. This activation compromises the integrity of the epithelial cell barrier and induces EMT through the p65/MMP-9 signaling pathway. These results highlight the crucial role of RBMX2 in M. bovis pathogenesis and underscore its potential as a therapeutic target for preventing infection-related complications.