Bacillus Calmette-Guérin ncBCG427 sRNA potentially enhances intracellular bacteria survival by inhibiting apoptosis and inflammatory responses

Abstract

Bacterial small RNAs (sRNAs) are non-coding RNA types that regulate target mRNA expression. In our previous research, we identified a novel sRNA, ncBCG427, that reduced the invasion of Mycobacterium smegmatis (M. sm) and altered its single-colony morphology. This study examined whether ncBCG427 produces similar effects in Bacillus Calmette-Guérin (BCG) and investigated the underlying mechanisms of its action. Overexpressing ncBCG427 in BCG (BCG_ncBCG427) increased bacterial growth, adhesion, and invasion in A549 cells, improving intracellular survival in THP-1 cells. In THP-1 cells, IL-1β, IL-6, TNF-α, phosphorylated p38, ERK, and p65 levels decreased, while p65 nuclear entry was inhibited, indicating ncBCG427 suppresses inflammation through the MAPK and NF-κB pathways. Additionally, the overexpression strain showed reduced levels of apoptosis. Transcriptomic analysis revealed 121 differentially expressed genes, identifying rpmB and JTY_2977 as key targets. Mutant strains with low expression of these genes (BCG_M-rpmB and BCG_M-JTY2977) exhibited enhanced growth, adhesion, and invasion in A549 cells, along with improved intracellular survival in THP-1 cells. A dual-luciferase reporter assay validated these genes as targets of ncBCG427. Furthermore, BCG_M-rpmB and BCG_M-JTY2977 decreased the expression of inflammatory cytokines and apoptosis, which were restored in complementary strains (BCG_C-rpmB and BCG_C-JTY2977). Our study demonstrated that ncBCG427 improved BCG intracellular survival by targeting rpmB and JTY_2977, suppressing inflammation and apoptosis via the MAPK and NF-κB pathways.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-026-05007-6.

Introduction

Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) is a leading cause of death worldwide, with approximately 10.7 million people developing TB in 2024 [1]. To address the threat of TB, the World Health Organization (WHO) launched the End TB Strategy in 2015 to eliminate TB by 2035. At the same time, the World Organization for Animal Health (WOAH) and the Food and Agriculture Organization of the United Nations (FAO) developed the 2030 Roadmap for Zoonotic Tuberculosis to support the WHO’s objective. Despite these initiatives, tuberculosis continues to be a significant cause of human mortality due to various factors: the global epidemic, the limited effectiveness of current treatments (which involve multi-drug therapy for over six months), the prevalence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains, low vaccine effectiveness, and insufficient safety profiles [2]. The lack of early and effective diagnostic technologies also contributes to the ongoing challenge. The pathogen resides within host macrophages, allowing it to evade the host’s innate and adaptive immune defenses, leading to intracellular survival and persistent infection.

Bacterial small RNAs (sRNAs) are a class of non-coding RNAs (ncRNAs) consisting of 40 to 400 nucleotides. Although these molecules are transcribed in cells, they do not code for proteins [3]. They play a vital role in numerous physiological processes and can be involved in both systemic and specific regulation of functions like virulence [4–6], metabolism [7], the inflammatory response [8], and assisting bacteria to adapt to and survive the internal environment of host cells [8, 9]. The advent of RNA sequencing (RNA-seq) has facilitated the discovery of numerous sRNAs in bacterial infections, including those caused by Mycobacterium species. For example, sRNA F6 can modulate the expression of essential chaperonins GroEL2 and GroES, which are important for the long-term survival and persistence of M. tb [10]. Similarly, sRNA MTS2823 has been shown to regulate the growth of multidrug-resistant strains within macrophages [11].

The inflammatory response is a defense mechanism that organisms develop against pathogenic microbial infections, trauma, allergies, and various other factors. Pathogenic mycobacteria have evolved complex strategies to evade cellular inflammatory responses. For instance, Rv1172c and Rv0309 have been shown to suppress the inflammatory response in host cells, thereby enhancing bacterial survival [12, 13]. In contrast, the sRNA SaaS can enhance bacterial virulence by stimulating the inflammatory response [10]. During M. tb infection, several key inflammatory molecules are involved. Interleukin-6 (IL-6) binds to its specific receptors, activating the MAPK and JAK-STAT signaling pathways, which leads to various immunological regulatory effects [14]. Likewise, tumor necrosis factor-alpha (TNF-α) regulates cellular inflammatory responses and apoptosis [15].

In our previous study, we utilized M. tb and BCG to infect THP-1 cells and identified a novel non-coding RNA, ncBCG427, which is 56 bp in length and located between JTY_3719 and JTY_3720. We demonstrated that ncBCG427 reduced bacterial invasion in M. sm expressing ncBCG427 [16]. This study aimed to confirm that ncBCG427 plays a similar role in BCG and to clarify the underlying mechanism. Our findings indicated that ncBCG427 enhanced the adhesion, invasion, and intracellular survival of BCG while inhibiting inflammation and apoptosis following BCG infection. This regulation occurs by targeting rpmB and JTY_2977, which affects the MAPK and NF-κB signaling pathways by preventing the entry of p65 into the nuclei.

Materials and methods

Bacterial strains, cells, and culture conditions

M. bovis BCG (Tokyo strain, ATCC 35737) was generously provided by Junyan Liu from Wuhan University. The recombinant vector pMV261-rrnBP and the plasmid pMV261-rrnBP-ncBCG427, which encodes ncBCG427, were previously constructed in our laboratory [16]. We developed the overexpression BCG strain (BCG_ncBCG427) and the control strain (BCG_Vector) by transforming BCG with pMV261-rrnBP-ncBCG427 and pMV261-rrnBP, respectively [17]. BCG was cultured to mid-log phase in 20 mL of Middlebrook 7H9 medium, supplemented with 10% oleic acid, albumin, dextrose, and catalase, as well as 0.05% Tween-80. The cultures were incubated at 37℃ for 2–4 weeks. The recombinant BCG strains were grown under the same media and conditions, with the addition of kanamycin at a concentration of 50 µg/mL as needed.

Insertion mutant strains (BCG_M-rpmB and BCG_M-JTY2977) were constructed in our previous work on constructing a mutant library [18]. Full-length rpmB (Gene ID: 7561025) and JTY_2977 (Gene ID: 7563837) were amplified from BCG genomic DNA using specific primers (rpmB, forward: 5’-ccgaagcttatgtccgcccgctgccaaat-3’ and reverse: 5’-ccaaagctttcacaggatcgccgccgttc-3’. JTY_2977, forward: 5’-aaactgcaggtggagcacggcaatccgca-3’ and reverse: 5’-cccaagctttcatgctgcgaggctgctga-3’). The target genes were then cloned into the pMV261 vector and electroporated into the mutant BCG strains to generate the complement strains BCG_C-rpmB and BCG_C-JTY2977.

A549 human lung adenocarcinoma cell line (A549 cells, ATCC# CRL-7909) and THP-1 human monocytic leukemia cell line (THP-1 cells, ATCC# TIB-202) were passaged and preserved in the laboratory. A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, HyClone) supplemented with 10% fetal bovine serum (FBS, Gibco). THP-1 cells were maintained in RPMI medium (HyClone) supplemented with 10% FBS. Cells were cultured at 37◦C in a humidified atmosphere with 5% CO₂. Cell types for the various experiments were chosen based on established protocols from the previous studies [19].

RNA extraction

Extracellular bacterial total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. For intracellular bacterial samples, bacteria were separated from eukaryotic cells at the designated infection time points, washed twice with RPMI-1640 medium, and then resuspended in GTC lysis solution. After rapid lysis, the preparation was centrifuged at 5000×g for 20 min, followed by an additional centrifugation at 10,000×g for 20 s after resuspension in GTC. Tween-80 was added and mixed for 2 min, followed by the addition of RNA-free water. Total RNA from intracellular bacterial samples was extracted using the FastRNA Blue Kit (MP Biomedicals), following the manufacturer’s instructions. The concentration of total RNA was measured using an ultraviolet spectrophotometer at a wavelength of 260/280 nm (Thermo).

Invasion and adhesion

Invasion and adhesion assays were performed using human type II alveolar epithelial cells A549. A total of 10⁵ cells were added to each well of a 24-well tissue culture plate. A549 cells were infected with live BCG at a multiplicity of infection (MOI) of 10 for 1 h at 37℃ for the invasion assay, and for 30 min at 4℃ for the adhesion assay. Following infection, the cells were washed three times with sterile 1×phosphate-buffered saline (PBS, Gibco) and incubated with 1 mL of DMEM containing 10% FBS. For the invasion assay, 100 µg/mL of gentamicin was also included in the complete medium. Infected cells were permeabilized by incubation with 0.025% Triton X-100 (Sigma-Aldrich) for 20 min at the specified time points. Afterward, cell lysates were serially diluted and plated onto 7H11 agar plates supplemented with 10% OADC. Bacterial colony-forming units (CFUs) were quantified after 3 weeks of incubation, with each sample plated in triplicate.

Assay of intracellular bacterial survival

Phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich)-differentiated THP-1 cells were infected with the BCG strain at a MOI of 10 for 2 h at 37℃. Following the infection, the cells were washed three times with sterile 1×PBS to remove extracellular bacteria. This time point was designated as 0 h, after which the cells were cultured in complete medium containing 100 µg/mL gentamicin for various durations (12, 24, 36, and 48 h). Subsequently, cell lysates were serially diluted and plated onto 7H11 agar plates supplemented with 10% OADC. Bacterial CFUs were quantified after 3 weeks of incubation, with each sample plated in triplicate.

Additionally, at various time points post-infection, cells and supernatants were separately collected via centrifugation for further analysis of inflammatory cytokines and inflammation-related signaling pathway molecules.

Proinflammatory cytokine detection

The expression levels of proinflammatory cytokines IL-1β (Cat MM-0181H1), IL-6 (Cat MM-0049H1), and TNF-α (Cat MM-0122H1) in cell culture supernatants were determined using ELISA kits (MEIMIAN) following the manufacturer’s instructions. Each sample was plated in triplicate.

Expression detection on critical molecules in signal pathways associated with inflammation

Western blot assays were employed to detect non-phosphorylated and phosphorylated p38, ERK, and p65. In brief, infected cells were harvested and lysed in radioimmunoprecipitation assay (RIPA, Sigma-Aldrich) lysis buffer for 30 min on ice. Proteins were then separated using 12% SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with 5% free-fat dry milk for 3 h at room temperature, followed by overnight incubation at 4℃ with primary antibodies. The primary antibodies used included anti-p38 (Cat no. 8690), anti-phosphorylated-p38 (Cat no. 4511), anti-extracellular signal-regulated kinase (ERK) (Cat no. 4695), and anti-phosphorylated ERK (Cat no. 4370), anti-p65 (Cat no. 8242), anti-phosphorylated-p65 (Cat no. 3033) (Cell Signaling Technology), along with a mouse polyclonal antibody recognizing β-actin as an internal reference (Proteintech, Cat no. 60008-1-Ig). After washing the membranes three times with TBS containing 0.05% Tween 20, they were incubated with secondary antibodies (HRP Goat Anti-Rabbit IgG, Abbkine, Cat A21020; HRP Goat Anti-Mouse IgG, Abbkine, Cat A21010) for 1 h at room temperature. Protein bands were visualized using an ECL kit, and protein expression levels were quantified using ImageJ software (Version 1.51j8).

Confocal microscopy was employed to analyze the entry of p65 into the nuclei. A549 cells were cultured overnight and subsequently transfected with a recombinant plasmid encoding p65-EGFP (Beyotime, Cat no. D2820). After 24 h post-transfection, the nuclei were stained with Hoechst (Beyotime, Cat no. C1022), and the cells were infected with the corresponding BCG strains. At various time points post-infection, spaced at 1 h intervals, the cells were observed using a confocal high-content cell imaging analysis system (PerkinElmer Life and Analytical Sciences Ltd).

Detection of apoptosis and reactive oxygen species levels

The flow cytometry was used to detect apoptosis levels of infected cells. The A549 cells with and without various BCG strains (BCG-WT, BCG_ncBCG427, BCG_M-rpmB, BCG_M-JTY2977, BCG_C-rpmB, and BCG_C-JTY2977) were digested with 10% trypsin, collected, and washed three times with PBS, they were dyed by Annexin V-FITC/PI (Vazyme, Cat no. A211) and DCFH-DA (Beyotime, Cat no. S0035S) and detected using CytoFLEX-LX (Beckman Coulter).

Bioinformatic prediction and confirmation

BCG_ncBCG427 and BCG_Vector were cultured to the mid-log phase in Middlebrook 7H9 medium for a duration of 2–4 weeks. Following this, RNA was extracted from both bacterial strains for transcriptomic analysis at Novogene. Three replicates were selected for each sample group.

RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) was employed to predict the secondary structure of sRNA. IntaRNA (http://rna.informatik.uni-freiburg.de/IntaRNA/Input.jsp) was utilized to analyze interactions between sRNA and mRNA. Gene Ontology (GO) enrichment analysis was conducted using DAVID (https://david.ncifcrf.gov/home.jsp), while KOBAS version 3.0 (http://kobas.cbi.pku.edu.cn) was used for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.

A dual luciferase reporter assay was used to confirm the interaction of ncBCG427 and the promoter of the 3’-untranslated region (UTR) of potential target genes. Briefly, the wild-type (WT) and mutants (mut) 3’-untranslated region (UTR) of potential target genes, which contains the predicted ncBCG427 binding sites, were cloned into the psiCHECK2 luciferase reporter vector (Promega) to obtain the constructs encoding 3’-UTR-luc of target genes. The constructs were cotransfected with ncBCG427 mimic or NC mimic into HEK 293T cells. A luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega) 24 h after cotransfection.

Statistical analyses

The assays and experiments were carried out in triplicate, with the data presented as means ± standard errors of the mean (SEMs) from the triplicates. Statistical analysis was conducted using GraphPad Prism software (Version 9.0), with Student’s t-test utilized for comparisons between two groups and analysis of variance (ANOVA) for comparisons among more than two groups. Statistical significance is indicated at four levels: non-significant (ns) p > 0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Results

ncBCG427 enhanced BCG adhesion, invasion, and survival

The growth of BCG_ncBCG427 and BCG_Vector was compared over time. From days 6 to 27 post-inoculation, BCG_ncBCG427 displayed significantly higher growth than BCG_Vector. However, from day 30 onward, BCG_ncBCG427 grew more slowly than BCG_Vector (Fig. 1C).

Fig. 1.

Adhesion, invasion, and survival ability of BCG_ncBCG427. A-B Adhesion and invasion assays of BCG strains were conducted using A549 cells infected with BCG strains at an MOI of 10. The cells were incubated at 4 °C for 30 min for the adhesion assay and at 37 °C for 1 h for the invasion assay. The quantities of intracellular BCG were determined using CFUs. A Adhesion. B Invasion. C-D Assessment of survival ability for BCG_ncBCG427. C The growth rate of BCG_ncBCG427 was evaluated by incubating in 7H9 medium at 37℃. D Intracellular survival ability in THP-1 cells. Student’s t-test and ANOVA were applied to assess the statistical significance of differences among the groups. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistically significant differences

Additionally, plate counting assays demonstrated that the number of BCG_ncBCG427 bacteria adhering to and invading A549 cells was significantly higher than that of BCG_Vector (p < 0.05) (Fig. 1A-B). Furthermore, the intracellular survival of BCG in THP-1 cells was assessed. At 36- and 48-hours post-infection (hpi), the intracellular levels of BCG_ncBCG427 were significantly greater than those of BCG_Vector (p < 0.05) (Fig. 1D). These results indicate that ncBCG427 significantly enhanced adherence and invasion in A549 cells while also promoting the intracellular survival of BCG within THP-1 cells.

ncBCG427 inhibited inflammation response through MAPK and NF-κB signaling pathways

To explore the regulatory role of ncBCG427 in the inflammatory response, a Western blot assay was performed to assess the phosphorylation levels of key factors in the NF-κB and MAPK pathways, specifically p38, ERK, and p65. Compared to THP-1 cells infected with BCG-WT, the phosphorylation levels of p38, ERK, and p65 in THP-1 cells infected with BCG_ncBCG427 were significantly reduced at 24 hpi (p < 0.01). Additionally, at 48 hpi, the phosphorylation levels of p38 and p65 were significantly decreased (p < 0.001) (Fig. 2A-D), indicating that ncBCG427 overexpression may be responsible for inhibiting the MAPK and NF-κB signaling pathways. Additionally, confocal high-content cell imaging analysis demonstrated that the entry of p65 into the nuclei of A549 cells infected with BCG_ncBCG427 was reduced compared to BCG-WT, as indicated by lower fluorescence intensity, further supporting the inhibition of the NF-κB signaling pathway (Fig. 2H, Fig. S3A).

Fig. 2.

Detection of inflammatory factors related to MAPK and NF-κB signaling pathways of BCG_ncBCG427. A-D The expression levels of p65, ERK, and p38, along with their phosphorylated proteins. E-G The protein expression of IL-1β, IL-6 and TNF-α. H Confocal microscopic analysis of the nuclear localization of BCG_ncBCG427. A549 cells were transfected with vectors encoding p65-EGFP, and the nuclei were stained with Hoechst 33,342. Scale bars, 5 μm. ANOVA was employed to assess the statistical significance of differences between groups. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistically significant differences

To confirm the inhibitory effect on the NF-κB/MAPK pathways, levels of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were measured after infection. Compared to BCG-WT, BCG_ncBCG427 significantly reduced the expression of these cytokines at 24 and 48 hpi (p < 0.05) (Fig. 2E-G), aligning with the observed inhibition in the NF-κB and MAPK pathways. These findings suggest that ncBCG427 could inhibit the cellular inflammatory response in BCG-infected THP-1 cells.

ncBCG427 inhibited cell apoptosis

Flow cytometry analysis revealed that A549 cells infected with BCG_ncBCG427 exhibited a significant reduction in apoptotic proportions compared to those infected with BCG-WT (p < 0.001) (Fig. 3A-B). Additionally, we evaluated the levels of reactive oxygen species (ROS) in these groups. The peak fluorescence intensity in BCG-WT-infected cells shifted to the right when compared to the peaks observed in both BCG_ncBCG427-infected and uninfected A549 cells, suggesting that the overexpression of ncBCG427 led to decreased ROS levels in the infected A549 cells (Fig. 3C).

Fig. 3.

Detection of apoptosis and reactive oxygen species levels. A Cell apoptosis was detected using flow cytometry at 24 hpi. B Total apoptotic proportions were analyzed across various groups. C Detection of ROS (reactive oxygen species) levels. ANOVA was employed to assess the statistical significance of differences between the groups. ***p < 0.001 indicates statistically significant differences

Bioinformatics analysis to predict the target genes

Transcriptome sequencing was performed to identify the target genes of ncBCG427. A total of 121 genes were found to be differentially expressed when comparing the BCG_ncBCG427 and BCG_vector strains (Fig. S2A-C). We initially selected six genes from this dataset for further validation based on their interaction regions with sRNA and rankings (Fig. S1A). Subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that rpmB is enriched in the ribosomal L28 protein, while JTY_2977 is associated with transposition (Fig. S2E-F). Notably, rpmB and JTY_2977 ranked relatively high among the six selected genes. Therefore, we decided to focus on rpmB and JTY_2977 for further experiments.

rpmB and JTY_ 2977 are the target genes of ncBCG427

To verify the target genes of ncBCG427, we utilized intaRNA to predict the interaction sites between ncBCG427 and its target genes (Fig. 4A). We then constructed wild-type and mutant dual fluorescence reporter plasmids based on these predicted sequences and validated their interactions through dual fluorescence reporting experiments. The results indicated that rpmB and JTY_2977 interacted with ncBCG427, as shown by a significantly lower Rluc/Fluc ratio in the wild-type groups compared to the control (p < 0.001 for rpmB and p < 0.01 for JTY_2977). In contrast, the mutant groups showed no significant differences from the control group (Fig. 4B), confirming that rpmB and JTY_2977 are target genes of ncBCG427. We then determined the genomic positions of these genes (Fig. 4C) and predicted their protein secondary and tertiary structures (Fig. 4D-E). Collectively, rpmB (124055–124339) and JTY_2977 (3260272–3260661) were identified as target genes of ncBCG427. Further analysis of the evolutionary context of these genes revealed that rpmB is widely present across various bacterial genomes, with its phylogenetic tree in mycobacterium depicted in (Fig. 4F). At the same time, JTY_2977 is conserved within mycobacterium and predominantly found in M. tb and its variants (Fig. 4G). Given that ncBCG427 regulated bacterial biological characteristics and host inflammatory responses, we aimed to investigate whether rpmB and JTY_2977 share similar regulatory mechanisms with ncBCG427 in subsequent experiments.

Fig. 4.

Structure prediction of rpmB and JTY_2977 along with their interaction with ncBCG427. A Interaction sites between rpmB, JTY_2977, and ncBCG427. B Dual-Luciferase reporter assay of rpmB and JTY_2977 interacting with ncBCG427. C Genomic locations of rpmB and JTY_2977. D-E Predictions of the secondary and tertiary protein structures of rpmB and JTY_2977. D rpmB. E JTY_2977. F-G Phylogenetic tree. F rpmB. G JTY_2977. ANOVA was employed to assess the statistical significance of differences among the various groups. **p < 0.01 and ***p < 0.001 indicate statistically significant differences

BCG_M-rpmB and BCG_M-JTY2977 enhanced adhesion, invasion, and intracellular survival

qPCR was conducted to confirm the expression levels of rpmB and JTY_2977 in two mutant strains, showing significant reductions in expression for both genes (Fig. S1B). The growth results indicated that the mutant strains, BCG_M-rpmB and BCG_M-JTY2977, exhibited markedly higher growth compared to BCG-WT between days 18 to 24 and days 18 to 27, respectively, after which their growth began to decline on day 36 (Fig. 5A-B). Furthermore, plate counting assays revealed that the bacterial adhesion (p < 0.01) and invasion (p < 0.001) capabilities of BCG_M-rpmB and BCG_M-JTY2977 were substantially more significant than those of BCG-WT. In terms of intracellular survival, the intracellular levels of BCG_M-rpmB and BCG_M-JTY2977 were also significantly higher than those of BCG-WT at both 24 h (p < 0.05) and 48 hpi (p < 0.05 for rpmB and p < 0.01 for JTY_2977). Overall, the adhesion, invasion, and intracellular survival abilities of BCG_M-rpmB and BCG_M-JTY2977 were significantly enhanced (Fig. 5C-E).

Fig. 5.

Adhesion, invasion, and survival abilities of BCG_M-rpmB and BCG_M-JTY2977. A-B Growth rates of BCG incubated in 7H9 medium at 37℃. A BCG_M-rpmB. B BCG_M-JTY2977. C-D Adhesion and invasion assays of BCG strains. A549 cells were infected with BCG strains at an MOI of 10 for 30 min at 4℃ for the adhesion assay and for 1 h at 37℃ for the invasion assay. The numbers of intracellular BCG were quantified using CFUs. C Adhesion. D Invasion. E Intracellular survival of mutant strains. ANOVA was used to assess the statistical significance of differences between the groups. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistically significant differences

BCG_M-rpmB and BCG_M-JTY2977 inhibited inflammation responses

The expression levels of rpmB and JTY_2977 in two complementary strains were significantly increased, as confirmed by qPCR (p < 0.001) (Fig. S1C). Based on our earlier findings regarding ncBCG427, we hypothesized that ncBCG427 might exert its anti-inflammatory effects by inhibiting the expression of its target genes, rpmB and JTY_2977, thereby impacting the key factor p65. To further validate this hypothesis, we examined the phosphorylation levels of p65 in the two mutant strains. We noted a significant decrease at 48 hpi (p < 0.001) (Fig. 6A-B), while the complementary strains exhibited an increase (p < 0.05) (Fig. 6H-I). Using a confocal high-content cell imaging analysis system, we discovered that both BCG_M-rpmB and BCG_M-JTY2977 inhibited the entry of p65 into the nucleus, whereas no significant inhibition was observed in the complementary strains, BCG_C-rpmB and BCG_C-JTY2977 (Fig. 6O, Fig. S3B). We also assessed the phosphorylation levels of ERK and p38. The results showed that the phosphorylation levels of p38 in both mutant strains were similar to those in BCG_ncBCG427, with significant reductions observed at 24 hpi (p < 0.01). Additionally, the phosphorylation level of ERK in the mutant strains was significantly decreased at 48 hpi (p < 0.001), although there was no significant difference for BCG_M-rpmB compared to BCG-WT at 24 hpi (Fig. 6A, C-D). For the complemented strains, except for BCG_C-JTY2977, which showed no significant difference in p38 phosphorylation levels compared to the control at 24 hpi, the phosphorylation levels of ERK and p38 in the two complemented strains were significantly elevated at both 24 and 48 hpi (p < 0.05) (Fig. 6H, J-K). Moreover, BCG_M-rpmB and BCG_M-JTY2977 significantly reduced the expression of IL-1β and TNF-α at both 24 and 48 hpi (p < 0.05) and significantly decreased IL-6 expression at 48 hpi (p < 0.01) (Fig. 6E-G). In contrast, both BCG_C-rpmB and BCG_C-JTY2977 significantly increased the expression of IL-6 and TNF-α at 24 hpi (p < 0.05), as well as IL-1β and TNF-α at 48 hpi (p < 0.01). Additionally, BCG_C-JTY2977 also elevated the expression of IL-6 at 48 hpi (p < 0.05) (Fig. 6L-N).

Fig. 6.

Detection of inflammatory factors related to MAPK and NF-κB signaling pathways of BCG mutant and complement strains. A-G mutant strains (BCG_M-rpmB and BCG_M-JTY2977). H-N complement strains (BCG_C-rpmB and BCG_C-JTY2977). A-D, H-K The expression of p65, ERK and p38 and their phosphorylated protein. E-G, L-N The protein expression of IL-1β, IL-6 and TNF-α. O Confocal microscopic analysis of nuclear localization of mutant strains. A549 cells were transfected with vectors encoding p65-EGFP, nuclei were stained with Hoechst 33,342. Scale bars, 5 μm. ANOVA was employed to assess the statistical significance of differences among the groups. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistically significant differences

BCG_M-rpmB and BCG_M-JTY2977 inhibited cell apoptosis

Flow cytometry was utilized to assess the apoptotic proportions of A549 cells, both uninfected and infected with various BCG strains: BCG-WT, BCG_M-rpmB, BCG_M-JTY2977, BCG_C-rpmB, and BCG_C-JTY2977. Compared to the BCG-WT infection group, both BCG_M-rpmB and BCG_M-JTY2977 significantly reduced apoptosis in A549 cells following infection. In contrast, the complementary strains, BCG_C-rpmB and BCG_C-JTY2977, increased the proportion of apoptotic cells relative to their respective mutant strains (Fig. 7A-B). Additionally, the analysis of ROS levels revealed that the peak fluorescence intensity of A549 cells shifted notably to the left after infection with the two mutant strains, indicating a reduction in ROS levels compared to cells infected with BCG-WT. Conversely, infection with the complementary strains resulted in a rightward shift in peak fluorescence intensity, suggesting an increase in ROS levels compared to the mutant strains. These findings indicate that BCG_M-rpmB and BCG_M-JTY2977 effectively lower ROS levels in A549 cells, while the complementary strains elevate these levels (Fig. 7C).

Fig. 7.

Detection of apoptosis and reactive oxygen species levels. A Cell apoptosis was detected by flow cytometry at 24 hpi. B Total apoptotic proportions were analyzed across different groups. C ROS (reactive oxygen species) levels detection. ANOVA was employed to assess the statistical significance of differences between different groups. ***p < 0.001 indicate statistically significant differences

Discussion

sRNAs, widely distributed among bacteria, vary in length and mode of action, playing crucial roles in various biological processes. They serve as key regulatory elements for gene expression in bacteria. A deeper exploration of the specific functions of sRNAs could significantly enhance our understanding of the fundamental aspects of life and improve strategies for preventing and controlling infectious diseases in both humans and animals [20, 21]. Despite their importance, research on the effects of sRNAs in mycobacteria—particularly regarding bacterial growth, virulence [22], and stress adaptability [23–25]—has been limited. Notable examples include sRNAs such as B11, MrsI, sRNA21, MTS1338, Mcr11, and 6 C [26, 27]. Given that TB is a critical zoonotic disease affecting both humans and animals, there is an urgent need to identify additional sRNA species and investigate their functions and underlying mechanisms. In previous work, we identified 349 sRNAs in M. tb and 490 sRNAs in BCG, with 96% of these being novel sRNAs. Furthermore, we identified this novel sRNA, ncBCG427, which was found to significantly reduce the invasion and alter the colony morphology of M. sm. Notably, ncBCG427 was initially discovered through sequencing of BCG and is absent from the M. sm. This prompted us to investigate whether ncBCG427 exerts similar functions in its native host, BCG. Interestingly, some effects were not consistent across the two mycobacterial species. While ncBCG427 decreased the invasion of M. sm, it significantly enhanced the invasion ability of BCG. Given that sRNAs typically function by regulating multiple target genes, this functional divergence may be attributed to genomic differences between M. sm and BCG. These findings also underscore that results obtained from model strains may serve primarily as references, a consideration that was central to the original rationale for this study. Through this study, we discovered that ncBCG427 enhanced the intracellular survival of BCG by inhibiting cellular inflammation and apoptosis through the MAPK and NF-κB signaling pathways.

The ncBCG427 targets rpmB and JTY_2977

Most sRNAs regulate the translation of one or more target mRNAs, resulting in either inhibition [28] or activation [29] of gene expression. For instance, the sRNA Qrr5 in Vibrio parahaemolyticus promotes self-aggregation and competitive symbiotic growth by modulating its target genes LysR, OmpA, and TraA [21]. Similarly, the sRNA RsmA in Pseudomonas aeruginosa interacts with retS mRNA, inhibiting RetS synthesis and thereby controlling key lifestyle changes, including clustering, rhamnolipid synthesis, and the production of purulent proteins and lipases [30]. Additionally, sRNA ReaL targets pqsC mRNA to enhance the synthesis of Pseudomonas quinolone signal in Pseudomonas aeruginosa [31]. In this study, we predicted and screened the target genes of sRNA ncBCG427 through RNA sequencing and bioinformatic analysis, identifying rpmB and JTY_2977 as candidate targets. The interaction between ncBCG427 and these target genes was subsequently validated using a dual fluorescence reporter assay. Furthermore, a series of experiments—including Western blot assays, ELISA, confocal high-content cell imaging, and flow cytometry—demonstrated similar changes in inflammation and apoptosis in cells infected with mutant strains of both target genes (BCG_M-rpmB and BCG_M-JTY2977) as well as in cells overexpressing ncBCG427 (BCG_ncBCG427). Additionally, the downregulation of both target genes was confirmed through qPCR analysis. Collectively, these findings provide strong evidence that rpmB and JTY_2977 are direct targets of ncBCG427.

The ncBCG427 inhibits inflammatory response and apoptosis

Inflammation serves as the body’s first line of defense against pathogen infections. When macrophages are infected with M. tb, they initiate the production of various cytokines, including IL-1β, IL-6, and TNF-α, to promote an immune response aimed at eliminating the pathogenic bacteria [14, 32]. These pro-inflammatory cytokines are regulated by both the MAPK and NF-κB signaling pathways. IL-1β, a critical member of the IL-1 family, plays a vital role in the defense against tuberculosis [33]. IL-6, on the other hand, activates both the MAPK and JAK-STAT signaling pathways through its receptors, exerting significant immunomodulatory effects [14]. TNF-α is another key pro-inflammatory cytokine that contributes to immunity against M. tb-infected cells and can be produced by various innate and adaptive immune cells. Recent studies have identified CD38 + CD27-TNF-α markers on M. tb-specific CD4 + T cells as reliable biomarkers for tuberculosis diagnosis [34]. The NF-κB signaling pathway is widely recognized for its essential role in the inflammatory response, given its multifaceted functions. During the invasion of foreign pathogens, NF-κB enhances phagosome fusion by facilitating lysosomal release within phagosomes and increasing the production of membrane transport molecules, thereby playing a core role in the pro-inflammatory response [35]. Moreover, studies have confirmed the interplay between the NF-κB and MAPK pathways concerning the regulation of IL-1β, IL-6, and TNF-α. For instance, the reduction of dihydrotestosterone (DHT) can upregulate both NF-κB and MAPK pathways, leading to increased levels of IL-1β, IL-6, and TNF-α [36]. Conversely, XBF treatment has been shown to inhibit the NF-κB and MAPK pathways, resulting in decreased levels of these pro-inflammatory cytokines [37]. These findings underscore the complex regulatory networks governing the inflammatory response and the importance of cytokines in combating infections like tuberculosis.

Apoptosis is a physiological form of cell death that can be triggered through the mitochondrial or death receptor pathways [38]. In the context of M. tb infection, macrophage apoptosis plays a crucial role in controlling bacterial growth. This process can eliminate the bacteria through direct antibacterial activity or by encapsulating them within apoptotic bodies, thereby facilitating the recruitment of new macrophages and dendritic cells to the site of infection [39]. ROS are generated in macrophages by the oxidase NOX2, which is composed of various subunits located on the mitochondrial membrane [40]. Upon pathogen invasion, ROS production in macrophages escalates rapidly, creating oxidative stress that oxidizes intracellular biomacromolecules within the bacteria, ultimately inhibiting their growth [41]. TNF-α can activate several signaling pathways, including NF-κB, JNK-MAPK, and p38 MAPK, through its receptors, thus influencing cellular functions and apoptosis [15]. Understanding these processes is critical for elucidating the mechanisms by which macrophages respond to M. tb infection and how they contribute to the overall immune response. While our findings in A549 cells reveal epithelial-specific responses to BCG infection, future studies using macrophage cell lines such as THP-1 would be valuable to determine whether similar regulatory mechanisms operate in phagocytes.

Following infection with M. tb, host cells initiate a range of defense mechanisms, including inflammatory responses and apoptosis, to combat and eliminate the pathogen. The interplay between inflammation and apoptosis has been highlighted in numerous studies, revealing how common cytokines and signaling pathways mediate these processes. For instance, M. tb can induce apoptosis in polymorphonuclear neutrophils by activating the p38 MAPK signaling pathway, which in turn leads to the generation of ROS [42]. Additionally, TNF-α is a critical cytokine that can activate multiple signaling pathways, including NF-κB, JNK-MAPK, and p38 MAPK, thus regulating both inflammatory responses and apoptosis [15]. Lipoprotein LpqH from M. tb has also been shown to interact with the Toll-like receptor 2 (TLR2), triggering mitochondrial apoptosis in macrophages [43]. This interaction underscores the role of specific bacterial components in modulating host cell death pathways in response to infection. Moreover, innovative therapeutic approaches have emerged, such as the development of a microgel system that can be stimulated by ROS in situ. This system has been demonstrated to significantly reduce myocardial cell apoptosis and mitigate inflammatory responses, thereby enhancing therapeutic outcomes in models of myocardial infarction [44]. Recent research has further illuminated the dual role of apoptotic cells in inflammation. Apoptotic cells can release specific anti-inflammatory metabolites that induce gene programs in neighboring cells, promoting a state of inflammatory inhibition [45]. Additionally, apoptotic cells can suppress the production of pro-inflammatory factors through the release of transforming growth factor-beta (TGF-β), which further contributes to the resolution of inflammation [46]. These findings collectively emphasize the intricate balance between inflammation and apoptosis in the host’s immune response to M. tb and highlight potential therapeutic avenues for modulating these processes to improve outcomes in infectious and inflammatory diseases.

Our study demonstrated that the ncBCG427 overexpression strain (BCG_ncBCG427) and two mutant strains (BCG_M-rpmB and BCG_M-JTY2977) effectively inhibited the expression of key pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Through the use of a confocal high-content cell imaging analysis system, we speculated that ncBCG427 inhibits the nuclear translocation of the NF-κB p65 subunit, which is a critical mediator of inflammatory responses. This inhibition likely disrupts the activation of the NF-κB signaling pathway, leading to a further decrease in the expression of associated inflammatory factors and ultimately resulting in reduced cellular inflammation. Additionally, ncBCG427 suppressed levels of reactive oxygen species (ROS), which are often implicated in promoting apoptosis and cellular stress. Taken together, these results support the conclusion that ncBCG427 plays a significant role in inhibiting both inflammatory responses and apoptosis. This dual action suggests that ncBCG427 could be a promising candidate for therapeutic strategies aimed at managing inflammatory diseases, providing a protective effect against excessive inflammation and cell death. Besides, the use of pathway inhibitors in studies of M. tb offers a promising strategy to obtain more direct mechanistic evidence, an approach we can pursue in future work to validate and build upon our current findings.

The ncBCG427 promotes intracellular mycobacterial survival

For intracellular bacteria such as Mycobacteria, the ability to survive within host cells is as critical as their survival outside the host. Macrophages serve as the first line of defense and are essential initial effector cells in the host’s response to M. tb infection. They play a pivotal role in inhibiting the growth and transmission of pathogenic bacteria through various innate immune responses [47, 48]. However, during prolonged interactions with host macrophages, M. tb has evolved several immune evasion strategies to counteract the innate immune responses of these cells. These strategies include a range of mechanisms influenced by gene regulation that allow the bacteria to persist and thrive despite host defenses [49, 50]. In our study, we demonstrated that ncBCG427 decreased inflammatory responses and apoptosis by targeting key signaling pathways, specifically the NF-κB, JNK-MAPK, and p38 MAPK pathways, through the modulation of cytokines such as IL-1β, IL-6, and TNF-α. ncBCG427 achieved this by influencing the expression of genes such as rpmB and JTY_2977. Additionally, we assessed the growth curve and intracellular survival of BCG_ncBCG427 through plate counting assays. Our findings revealed that ncBCG427 not only increased the growth rate of the bacteria but also enhanced their intracellular survival during macrophage infection. This indicated that ncBCG427 promoted bacterial survival by inhibiting the cellular inflammatory response and apoptosis, thereby fostering a more favorable environment for bacterial persistence within host cells. Overall, these results underscore the potential of ncBCG427 as an important factor in enhancing the survival of Mycobacterium species within macrophages by modulating host immune responses, which could have implications for understanding M. tb pathogenesis and developing therapeutic strategies.

Conclusion

The ncBCG427 enhanced BCG adhesion, invasion, and intracellular bacterial survival by targeting rpmB and JTY_2977 to inhibit the inflammatory response and apoptosis through suppression of the phosphorylation of p38, ERK, and p65, as well as the nuclear entry of p65. (Fig. 8)

Fig. 8.

Proposed mechanisms of ncBCG427 regulation of BCG

Authors’ contributions

AG designed the study, KZ, ZD, CW, ZW, and HX conducted the experiments, KZ, CW, and HX were responsible for data organization and analysis, KZ wrote the initial draft of the article, and IS, YC, SL, SH, and AG edited and revised the article.

Funding

This research was funded by China Agriculture Research System of MOF and MARA (#CARS 37), National Key Research and Development Program of China (#2021YFD1800403) and Special Project for Enhancing Research Strength of Qinghai University (#2026KTST04).

Data availability

The sequencing data used in this study has been uploaded to the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/info/seq.html, GSE277118).

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.