Involvement of Mycobacterium smegmatis small noncoding RNA B11 in triacylglycerol accumulation and altered cell wall permeability

doi:10.21203/rs.3.rs-4810858/v1

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Involvement of Mycobacterium smegmatis small noncoding RNA B11 in triacylglycerol accumulation and altered cell wall permeability

https://doi.org/10.21203/rs.3.rs-4810858/v1

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Background Mycobacterium tuberculosis (M. tuberculosis) is known to causesevere lung disease in patients.Pathways involving triacylglycerol (TAG) accumulationare thought to play a crucial regulatory role in bacterial growth and metabolism. Despite this understanding, little is known about the biological functions and regulatory mechanisms of small RNAs in M. tuberculosis. Mycobacterium smegmatis (M. smegmatis), a type of Mycobacterium, serves as a model organism to investigate the molecular, physiological, and drug resistance features of M. tuberculosis.

Results In this study, we demonstrated that overexpression of B11 significantly affects bacterial growth and colony morphology, increases antibiotic sensitivity and sodium dodecyl sulfate (SDS) surface stress, decreases intracellular survival, and suppresses cytokinesecretion in macrophages. Transcriptomic and lipidomic analyses revealed a metabolic downshift in the B11 overexpression strain, characterized by reduced levels of TAG. Furthermore, transmission electron microscopy showed that the B11 overexpression strain exhibited decreased cell wall thickness, leading to reduced biofilm formation and altered cell wall permeability. Additionally, we observed that B11 regulated certain target genes but did not directly bind to proteins.

Conclusions Taken together, these findings suggest that B11 plays important roles in Mycobacterium survival under antibiotic and SDS stresses, TAG accumulation, and contributes to antibiotic sensitivity through altered cell wall permeability.

Mycobacterium smegmatis

B11

triacylglycerol

cell wall permeability

biofilms

Infections caused by mycobacterium are major public health problems globally.

Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis (TB), infects one-third of the world's population [1].The unique cell wall structure of mycobacterium contributes to its resilience against the host immune system. Multiple cell wall lipids enhance impermeability, intrinsic resistance, persistence within macrophages, virulence, and adaptability to the host environment during infection [2]. Understanding the implications of outer membrane construction for cellular fitness and survival can offer insights into drug development and TB treatment [3].

Mycobacterium can adapt to changing environments and persist within the host due to its capacity to remodel metabolism and resist antibiotics [4]. Triacylglycerol (TAG), a glycerol triester with fatty acids, serves as a major carbon and energy source for M. tuberculosis during prolonged infection [5]. TAG accumulates in two forms: synthesized and stored in the bacterial cytoplasm, and present in the mycobacterial cell wall [6]. Cytoplasmic TAG in intracytoplasmic lipid inclusions (ILIs) confer phenotypic drug tolerance and promote pathogen survival [7][8]. Various factors, including hypervirulent M. tuberculosis strains, lipid-rich diets, and environmental stresses, induce ILI formation in mycobacteria [9–11]. Altered biofilms associated with reduced mycolic acid wax ester and long-chain TAG levels play crucial roles in adaptive immune pressure and non-replicating persistence [12].

Small RNAs (sRNAs), typically 50–300 nt in length, are essential molecules involved in regulatory processes [13]. While extensively studied in gram-negative bacteria, knowledge of sRNA functions and regulatory mechanisms in high G + C bacteria, such as M. tuberculosis, remains limited [14, 15]. As novel sRNAs are discovered and their biological functions elucidated, their role as key post-transcriptional regulators of gene expression becomes increasingly evident.

B11 (ncRv13660c, MTS2822, 6C) is approximately 95 nucleotides long and is situated in the intergenic region between Rv3660c and Rv3661, exhibiting a conserved secondary structure featuring two C-rich loops [16]. Its structure suggests a potential role as a structural or protein-binding RNA [16]. Initially identified in 2009 by Arnvig et al., B11 demonstrates increased expression in response to H₂O₂ [17]. While overexpression of B11 in M. tuberculosis proves lethal to the bacterium, its overexpression in Mycobacterium smegmatis (M. smegmatis) leads to slow growth [18]. Disruption of B11 in Mycobacterium kansasii correlates with alterations in colony morphology and biofilm formation [19], while its absence in M. abscessus results in heightened virulence and pro-inflammatory immune signaling [20]. In M. tuberculosis, B11 binds to panD and dnaB mRNA via C-rich loops, implicating it in various cellular processes [14], suggesting a potential role in cell division regulation. However, further comprehensive research on B11 is warranted to elucidate its biological functions and regulatory processes fully.

M. smegmatis shares over 2000 homologous genes with M.tuberculosis [21]. Its unique cell wall structure is akin to that of M. tuberculosis and various other mycobacterial species [22]. Consequently, M. smegmatis has been employed as a model organism to investigate the molecular, physiological, and drug resistance mechanisms of M.tuberculosis. In this study, we conducted a comprehensive analysis of M.smegmatis B11. We found that B11 overexpression affects bacterial growth and colony morphology, increases antibiotic sensitivity and sodium dodecyl sulfate (SDS) surface stress, decreases intracellular survival, and suppresses cytokine secretion in macrophages. Through RNA-seq and lipidomics analysis, we characterized the regulatory and metabolic pathways modulated by B11, particularly affecting TAG accumulation. Additionally, transmission electron microscopy (TEM) revealed a reduction in cell wall thickness associated with B11 overexpression, along with diminished biofilm formation and altered cell wall permeability. Furthermore, we observed that while B11 regulates certain target genes, it does not directly bind to proteins.

Mutant isolation and phenotype analysis.

Previously, [14] reported that the C-rich loops of 6C sRNA are required for growth inhibition. To investigate the functions of B11, the RNAfold program was employed to predict the secondary structure of M. tuberculosis B11 (Fig. 1A). To assess the criticality of these C-rich loops for function, mutations were introduced in this region of L2 and L3 (C-to-A conversion at C-rich sequences) (Fig. 1B). Results indicated that Ms_B11 exhibited a smooth phenotype on a solid medium (Fig. 1C). Furthermore, the Ms_B11 strains displayed significantly decreased sliding motility (Fig. 1D). Compared to the control strain, Ms_B11 began to show reduced growth (Fig. 1E). SEM examination revealed that Ms_B11 strains were significantly elongated compared to the control cells (Fig. 1F). Additionally, Ms_B11 strains exhibited significantly increased cell lengths (2.01 ± 0.85 µm) compared to wild-type strains (1.32 ± 0.56 µm) (Fig. 1G). We also observed that the cell lengths were partially recovered in the Ms_B11 L2 and Ms_B11 L3 strains (1.67 ± 0.73 µm, 1.64 ± 0.59 µm, respectively) (Fig. 1G).

Overexpression of B11 enhances antibiotic sensitivity and SDS surface stress.

Subsequently, we examined the susceptibility to vancomycin and linezolid, which target bacterial cell wall assembly and protein synthesis, respectively. The B11 overexpression strain exhibited decreased survival rates when exposed to high concentrations of vancomycin (5 µg/mL) and linezolid (1 µg/mL) compared to the empty vector strain (P < 0.05; Fig. 2A, B). Furthermore, when subjected to SDS surface stress, the B11 overexpression strain displayed reduced survival rates after exposure to 0.05% SDS for 1 and 5 h (Fig. 2C) compared to the empty vector control. Similar trends were observed with recombinant strains Ms_B11 and Ms_vc incubated with 0.05% SDS at varying intervals: the bacterial count in all recombinant strains exposed to 0.05% SDS decreased rapidly, with the B11 overexpression strain showing heightened sensitivity to SDS compared to the empty vector control (Fig. 2D).

Overexpression of B11 reduces intracellular survival and suppresses cytokine secretion in macrophages.

To assess whether B11 overexpression affects M. smegmatis survival within host cells, we infected THP-1-derived macrophages with the B11 overexpression and control strains. The B11 overexpression strain exhibited decreased intracellular survival during THP-1-derived macrophage infection (Fig. 3A). To evaluate the impact of B11 overexpression on pro-inflammatory cytokines in macrophages, the levels of TNF-α, IL-1β, and IL-6 were measured by ELISA. The supernatant of THP-1-derived macrophages infected with the B11 overexpression strain displayed lower levels of TNF-α (P = 0.002) (Fig. 3B), IL-1β (P = 0.02) (Fig. 3C), and IL-6 (P = 0.03) (Fig. 3D) compared to those infected with the empty vector control.

B11 modulates TAG accumulation in M. smegmatis.

To elucidate the regulatory and metabolic alterations induced by B11 overexpression in M. smegmatis, we conducted RNA-seq and lipidomics analyses. A total of 1,300 DEGs were identified in the B11 overexpression strain, comprising 785 upregulated and 515 downregulated genes (Fig. 4A, Supplementary Table 2). Gene Ontology analysis revealed significantly upregulated genes involved in DNA replication (BP), integral membrane component (CC), and NADH dehydrogenase (ubiquinone) activity (MF) in the B11 overexpression strain (Fig. 4B). Conversely, significantly downregulated genes were associated with cellular aromatic compound metabolic processes (BP), cytoplasmic localization (CC), and ATP binding (MF) (Fig. 4C). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that glycerolipid metabolism was predominantly enriched by upregulated genes in Ms_B11 strains (Fig. 4D), while mismatch repair pathways were most enriched by downregulated proteins (Fig. 4E). Additionally, numerous metabolic enzymes involved in glycerolipid metabolism were accumulated (Fig. 4F), underscoring the importance of this pathway in non-replicating persistence crucial for Mtb survival and re-growth.

Furthermore, comparative lipidomic analyses were conducted to assess differences in lipid profiles between Ms_B11 and Ms_vc strains. Among 10,967 total detected events, 103 met stringent criteria (VIP > 1, P < 0.05) (Supplementary Table 4). Notably, a distinct pattern emerged, with 34 events exclusively associated with TAG species (Fig. 5A). Heat map visualization of normalized concentration revealed differential abundances of metabolites (Fig. 5B). Individual TAG species, differing in length and unsaturation, were detected at significantly reduced levels in both Ms_B11 and Ms_vc strains (Fig. 5C). Subsequently, we investigated cell envelope ultrastructure and lipid inclusions in recombinant strains. M. smegmatis cells containing the B11 antisense overexpression vector exhibited ILIs (Fig. 6A, C). The proportion of cells with ILI profiles was more than halved in Ms_B11 compared to wild-type strains (Fig. 6D).

Overexpression of B11 alters cell envelope structure and biofilm formation

To delve deeper into the impact of B11 on bacterial cell envelopes, we examined the ultrastructural features of wild-type (WT) and recombinant strains using TEM. The cell envelope exhibited a triple-layered structure comprising the electron-dense outer layer, electron-transparent layer (ETL), and peptidoglycan layer (PGL) (Fig. 6B). Notably, the ETL thickness differed significantly between Ms_B11 (10.27 ± 5.05 nm) and wild-type strains (6.38 ± 2.89 nm), with the B11 overexpression strain showing a marked increase (12.46 ± 3.07 nm) (Fig. 6E).

Given the association between biofilm formation and cellular lipid profiles reported in various studies [6], we explored whether B11 overexpression and reduced TAG accumulation impact biofilm formation. The Ms_B11 strain exhibited significantly diminished biofilm growth and less structured pellicle compared to the wild-type strain, a trend also observed in the Ms_B11 L2 and Ms_B11 L3 strains (Fig. 7A). Consistently, crystal violet staining revealed a notable decrease in biofilm signal intensity in the B11 overexpression strain relative to wild-type strains (Fig. 7B).

To assess whether B11 overexpression affected cell wall permeability in M. smegmatis, we measured the accumulation rates of fluorescence dyes, EtBr, and Nile Red in Ms_B11 and Ms_vc strains. EtBr accumulation was significantly higher in the Ms_B11 strain compared to the control strain (Fig. 7C). Moreover, Nile Red dye, a lipid stain, exhibited increased uptake in the B11 overexpression strain relative to the control strain (Fig. 7D).

B11 indirectly modulates target genes.

The functional mechanisms of noncoding RNAs often involve direct and indirect interactions with RNA-binding proteins. Therefore, we conducted RNA pull-down assays in B11-overexpressing M. smegmatis strains using both control and B11 probes. Mass spectrometry identified 49 proteins (emPAI > 1) associated with B11, including accC, rpsB, hspX, SecA, and MSMEG_6518 (Fig. 8A, Supplementary Table 3). Combining transcriptome sequencing data, we observed significant upregulation and downregulation (P < 0.05) of a group of genes in the B11-overexpressed strain (Fig. 8B). To validate some of these expression changes, qRT-PCR was performed on RNA from the recombinant strain. The relative expression levels of hspX were significantly upregulated compared to the control group, while the expression of rpsB, MSMEG_6092, and rplJ was significantly downregulated (Fig. 8C). Further confirming the direct interaction, three recombinant M. smegmatis strains, Ms_hspX, Ms_rplJ, and Ms_6092 strains, were successfully constructed (Fig. 8D). However, RNA pull-down results demonstrated that B11 did not directly bind to these three proteins (Fig. 8E).

A pivotal aspect of TB pathogenesis is M. tuberculosis's ability to withstand diverse environmental stressors. Regulatory mechanisms triggered by stress play crucial roles in bacterial survival processes, including colony morphology, physiological adaptations, virulence, and antibiotic resistance. In most bacteria, survival mechanisms, such as the unique cell wall structure of mycobacteria acting as a permeable barrier, are tightly regulated at multiple levels. However, only a few reports have elucidated the physiological role of sRNAs, especially concerning survival under stressful conditions. In this study, we conducted a comprehensive investigation of B11 in M. smegmatis. B11 influenced cell wall morphology and TAG accumulation, potentially accounting for the observed growth and colony morphology changes in Ms_B11. The distinctive cell wall structure serves as a protective barrier against environmental stresses such as surface stress SDS and antibiotics. As anticipated, the recombinant Ms_B11 phenotype exhibited greater sensitivity to SDS surface stress, vancomycin, and linezolid compared to Ms_vc, suggesting a role for B11 in stress response.

According to previous studies, B11 appears to play a role in growth and colony morphology in bacteria, including M. tuberculosis, M. smegmatis [17], and M. kansasii [19]. Here, we utilized M. smegmatis mc²155 as our model to gain insights into the cellular function of B11. Our findings are consistent with previous reports: the expression of B11 resulted in slow growth and altered colony morphology, along with changes in cell wall-associated properties. The transition from rough to smooth colony morphology is closely associated with bacterial biofilm formation [23]. Congo red, an amphiphilic dye that binds to lipoproteins, was employed to assess the overall hydrophobicity of the mycobacterial cell wall [24]. Cell wall hydrophobicity is intricately linked to bacterial aggregation and biofilm formation. In our study, Congo red staining revealed significant differences between the B11 overexpression strains and the control strains. The altered Congo red staining of Ms_B11 colonies could also account for the impaired biofilm formation, which was further confirmed in a liquid medium. The abnormal Congo red staining and reduced biofilm formation of Ms_B11 colonies suggest that overexpression of B11 may inhibit biofilm formation.

Our further investigations into the cell lipid profiles revealed significant alterations in TAG accumulation in B11-overexpressed strains, indicating changes in ILI accumulation and cell wall integrity. TAG serves as the primary energy reservoir in Mycobacterium, accumulating in large quantities within cytoplasmic ILIs [6]. Here, we have confirmed that B11 can decrease TAG accumulation in M. smegmatis. Additionally, previous studies have reported substantial amounts of TAG in the outer membrane of M. smegmatis [25]. Our ultrastructural analysis revealed cell wall defects in B11-overexpressed strains, characterized by a significant reduction in the ETL, suggesting a potential impact on TAG accumulation within the cell wall. Prior research has underscored the significance of TAG in antibiotic tolerance and survival within lipid-rich environments [6, 26]. Specifically, ILI accumulation has been associated with phenotypic drug tolerance [27]. Consistent with these findings, our results demonstrate increased susceptibility of B11-overexpressed M. smegmatis to vancomycin and linezolid, indicative of reduced TAG accumulation. Moreover, we observed decreased intracellular survival of B11-overexpressed strains during macrophage infection. We provided evidence indicating that B11 overexpression leads to suppression of cytokine secretion, including TNF-α, IL-1β, and IL-6, in macrophages. While cytoplasmic TAG within ILIs may promote pathogen survival during dormancy and reactivation [6], the function of TAG accumulating on the cell wall remains unclear. Further investigation is warranted to elucidate the potential relationship between TAG accumulation and resistance or survival mechanisms. In summary, our findings suggest that B11 overexpression impacts TAG accumulation in M. smegmatis, influencing antibiotic tolerance and survival during macrophage infection.

Furthermore, our study revealed that B11 impacts cell wall permeability. The uptake of EtBr and Nile red has been utilized to assess mycobacterial cell wall permeability [24]. Previous research has linked the uptake of EtBr and Nile red to the lipid composition of the cell wall [28]. Here, we demonstrated for the first time, to our knowledge, that B11 alters cell wall permeability, evidenced by increased uptake of EtBr and Nile red. SDS serves as a valuable tool to investigate perturbations in the cell membrane/cell wall [29]. Notably, the survival capacity of Ms_B11 under SDS exposure was significantly diminished compared to control strains, indicating a cell wall defect in Ms_B11. Studies have underscored the significant impact of cell membrane permeability on drug penetration [30]. Decreased permeability of the bacterial cell wall can hinder antibacterial drug penetration [31]. Our experimental findings revealed that the B11 overexpression strain exhibited reduced survival following exposure to high concentrations of vancomycin and linezolid compared to the empty vector strain. This observation, suggesting a role for B11 in resistance to these two drugs, is particularly notable. Consistently, previous reports have indicated that B11 deletion mutants led to increased resistance to linezolid in M. abscessus [20]. Collectively, our results indicate that B11 influences survival under various in vitro stresses, including surface stress SDS and exposure to multiple antibiotics. Consequently, bacterial stress responses may contribute to the development of antibiotic resistance in bacteria exposed to stressful conditions [32]. However, a more comprehensive understanding of the impact of mycobacterial stress on antibiotic resistance warrants further investigation.

Additionally, our findings suggest that B11 negatively regulates certain genes but does not directly bind to proteins. It is established that sRNAs can modulate gene expression by directly binding to mRNA sites. Previous studies have reported that the C-rich loops of 6C sRNA base-pair with panD mRNA independently of RNA chaperones, although G-rich stretches were not identified in other validated targets (MSMEG 0408 and mce1F) [14]. Further research is required to elucidate whether novel classes of RNA chaperones modulate the activity of B11.

In summary, our study highlights the impact of B11 overexpression on M. smegmatis, revealing alterations in bacterial growth, colony morphology, and the inhibition of biofilm formation. Additionally, the observed reduction in triglyceride accumulation due to B11 overexpression correlates with changes in cell wall permeability and thickness, likely contributing to the observed phenotypic tolerance to vancomycin and linezolid. However, the precise function of the accumulated TAG on the cell wall remains unclear, presenting a promising avenue for potential drug development targets.

Bacterial strains and growth conditions

The research utilized M. smegmatis mc²155 and Escherichia coli DH5α strains. M. smegmatis mc²155, overexpressing strains, and mutant strains were cultured in a Middlebrook 7H9 medium supplemented with 0.2% glycerol, 0.05% Tween 80, and 50 mg/mL of kanamycin when necessary. A Middlebrook 7H10 medium containing 2% glycerol was employed for the cloning of M. smegmatis mc²155. E. coli DH5α strains were cultured in a Luria–Bertani (LB) medium supplemented with 50 mg/mL of kanamycin when required. All cultures were incubated at 37°C with shaking at 150 rpm.

Molecular cloning and plasmid construction

Supplementary Table 1 provides details of the bacterial strains, plasmids, and primer sequences used for cloning. Briefly, B11 and its mutants were cloned into pMV261 at the HindIII and NdeI sites. Genes from M. smegmatis mc²155 were PCR-amplified, digested with HindIII and BamHI, and then cloned into pMV261 with an N-terminal three-Flag tag. The resulting plasmids were electroporated into M. smegmatis mc²155 as previously described. DNA sequencing was conducted to confirm all constructs. The expression of proteins in recombinant strains was analyzed via western blotting.

qPCR analysis

Cells were collected and lysed using Fastprep-24 (MP Biomedicals) following the manufacturer’s instructions. Total RNA was isolated via phenol-chloroform extraction and ethanol precipitation. Subsequently, total RNA was reverse-transcribed to cDNA using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech). The mRNA expression of selected genes was analyzed using the same qPCR Master Mix. Mean ± standard error of mean values were calculated from three independent experiments, and significant differences were determined using Student’s unpaired t-test. The primer sequences utilized in this study are listed in Supplementary Table 1.

Western blot analysis

The expression of Flag-tagged HspX, RplJ, and MSMEG_6092 was verified using western blotting with a mouse anti-Flag-tag monoclonal antibody (Beyotime, China) as the primary antibody and an HRP-conjugated goat anti-mouse IgG antibody (Beyotime, China) as the secondary antibody. Lysates of recombinant strains were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and proteins were transferred on to polyvinylidene fluoride membranes. The membranes were blocked and then incubated with diluted primary antibodies. Subsequently, membranes were incubated with an HRP-labeled anti-rabbit IgG antibody (Beyotime, China), and the signal was developed using the Bio-Rad ChemiDoc XRS + instrument and image software.

Scanning electron microscopy

Scanning electron microscopy (SEM) was performed according to our published procedure [33]. Briefly, cells were collected and fixed with 2.5% glutaraldehyde overnight at 4°C. Sequential ethanol dehydrations were performed in a graded series, followed by critical point drying using a Hitachi HCP-2 Critical Point Dryer (Hitachi High-Technologies Corp., Tokyo, Japan). After drying, the samples were sputter-coated, and ultrastructure examination was conducted using a Zeiss Ultra55 electron microscope.

TEM

TEM was performed according to our published procedure. Cells were harvested, fixed, and dehydrated in a graded ethanol series, then embedded in Spurr resin. Thin sections were examined using a Tecnai Spirit (FEI) transmission electron microscope. Lipid inclusions in the intracytoplasmic space were regularly detected. Cell wall thickness was analyzed using ImageJ software.

Phenotypic analysis

Growth curve analysis, sliding motility assay, Congo Red assay, and biofilm assay were performed as previously described [33]. Bacterial cultures were grown to the exponential phase (OD = 0.2–0.4 at 600 nm). The optical density at 600 nm (OD₆₀₀) was measured at intervals of 3 h to obtain growth curves. M. smegmatis mc²155 recombinant strains were cultured in a LB medium supplemented with 100 mg/mL Congo Red (Sigma), a Sauton liquid medium, and a 7H9 medium supplemented with 0.3% agar, respectively, and phenotypes were observed.

Spot tests

Recombinant strains were cultured to exponential phase and adjusted to an OD₆₀₀ of 0.2. Ten-fold serial dilutions of recombinant strains were spotted onto 7H10 agar containing the indicated concentrations of SDS (0.05%), vancomycin (5.0 µg/mL), and linezolid (µg/mL).

Survival curves

Survival curve analysis was conducted as previously described [34]. Recombinant strains were treated with antibiotics and SDS at 37°C for various durations. Bacterial survival was quantified by colony formation on drug-free agar.

Macrophage infections and cytokine assays

The human leukemia monocytic cell line (THP-1) was utilized for this study. THP-1 macrophages were infected with recombinant strains at a multiplicity of infection (MOI) of 10:1. For the intracellular bacterial survival assay, cells were washed, lysed, serially diluted, and inoculated onto 7H10 agar. The supernatant of each culture was collected and analyzed for cytokine levels using an enzyme-linked immunosorbent assay (ELISA) kit (Beyotime).

RNA-Seq analysis

Total RNA was extracted from recombinant strains and used for sequencing library construction. The Ultra-™Directional RNA Library Prep Kit for Illumina (NEB) was employed according to the manufacturer’s instructions. The final library products were purified and assessed using an Illumina second-generation high-throughput sequencing platform at Allwegene Technology Inc., Beijing.

RNA pull-down

The full-length sense RNA of B11 was biotin-labeled using the Pierce™ RNA 3′ End Desthiobiotinylation Kit (BersinBio). Pull-down assays were performed by incubating biotin-labeled RNA with cell lysates using the RNA pull-down Kit (BersinBio). The bound proteins were subjected to mass spectrometry and Western blotting analysis.

Lipidomics analysis

Lipidomics analysis was conducted following our previously published protocol [33]. In brief, lipid extraction was performed using a chloroform/methanol solution (2:1, v:v) and an ultrasonic cleaner. The supernatant was then dried using N₂ and dissolved in chloroform/methanol solution (2:1, v:v). Lipid samples were processed and analyzed by Biotree Biotech (Shanghai, China). LC-MS/MS analyses were performed using a Dionex UltiMate 3000 (UHPLC)–Thermo Orbitrap Elite. Raw data were converted to the mz.data format using Agilent MassHunter Qualitative Analysis B.08.00 software (Agilent Technologies, United States), and further processed using the XCMS program. Multivariate analysis was performed using the SIMCA16.0.2 software package (Umetrics, Umea, Sweden).

Ethidium bromide and Nile red uptake assay

The ethidium bromide (EtBr) and Nile red uptake assay were conducted according to a previously established protocol [35]. Recombinant strains were cultured to exponential phase and adjusted to an OD₆₀₀ of 0.8. Then, 200 µL of bacterial suspension was added in triplicate to a 96-well black fluoroplate and treated with EtBr and Nile red dye to a final concentration of 2.0 µg/mL and 20 µM, respectively. The fluorescence intensity of accumulated dyes was measured on a BioTek Synergy HIM microplate reader (excitation: 544 nm, emission: 590 nm).

M. tuberculosis

Mycobacterium tuberculosis

tuberculosis

M. smegmatis

Mycobacterium smegmatis

TAG, triacylglycerol

SDS

sodium dodecyl sulfate

ILIs

intracytoplasmic lipid inclusions

sRNAs

Small RNAs

TEM

transmission electron microscopy

ETL

electron-transparent layer

PGL

peptidoglycan layer.

Funding

This study was supported by Dongguan Science and Technology of Social Development Program (grant number 20211800904782), Guangdong Provincial Clinical Research Center for Tuberculosis (No. 2020B1111170014 ), the Science and Technology Planning Project of Guangdong Province, China (No. 2021B1212030003), the Science and Technology Program of Guangzhou, China (No. 202201011764), and the Infectious Disease Prevention and Control of the National Science and Technique Major Project (No. 2018ZX10715004-002).

Author Contribution

ZW, XC, JL and HW designed the experiments. ZW, WL, QT, YC, XL JH and HL performed the experiments, analyzed the data, and interpreted the results. ZW and QT wrote the manuscript. The author(s) read and approved the fnal manuscript.

Data Availability

The RNA-seq and lipidomics data has been submitted to the Sequence Read Archive (SRA) and OMIX database with the bioProject accession numbers PRJNA1097297 and PRJCA028533, respectively.

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Involvement of Mycobacterium smegmatis small noncoding RNA B11 in triacylglycerol accumulation and altered cell wall permeability

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Abstract

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Overexpression of B11 alters cell envelope structure and biofilm formation

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Materials and methods

Bacterial strains and growth conditions

Molecular cloning and plasmid construction

qPCR analysis

Western blot analysis

Scanning electron microscopy

TEM

Phenotypic analysis

Spot tests

Survival curves

Macrophage infections and cytokine assays

RNA-Seq analysis

RNA pull-down

Lipidomics analysis

Ethidium bromide and Nile red uptake assay

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