-
The Journal of Biological Chemistry May 2024Mycobacterial genomes encode multiple adenylyl cyclases and cAMP effector proteins, underscoring the diverse ways these bacteria utilize cAMP. We identified universal...
Mycobacterial genomes encode multiple adenylyl cyclases and cAMP effector proteins, underscoring the diverse ways these bacteria utilize cAMP. We identified universal stress proteins, Rv1636 and MSMEG_3811 in Mycobacterium tuberculosis and Mycobacterium smegmatis, respectively, as abundantly expressed, novel cAMP-binding proteins. Rv1636 is secreted via the SecA2 secretion system in M. tuberculosis but is not directly responsible for the efflux of cAMP from the cell. In slow-growing mycobacteria, intrabacterial concentrations of Rv1636 were equivalent to the concentrations of cAMP present in the cell. In contrast, levels of intrabacterial MSMEG_3811 in M. smegmatis were lower than that of cAMP and therefore, overexpression of Rv1636 increased levels of "bound" cAMP. While msmeg_3811 could be readily deleted from the genome of M. smegmatis, we found that the rv1636 gene is essential for the viability of M. tuberculosis and is dependent on the cAMP-binding ability of Rv1636. Therefore, Rv1636 may function to regulate cAMP signaling by direct sequestration of the second messenger. This is the first evidence of a "sponge" for any second messenger in bacterial signaling that would allow mycobacterial cells to regulate the available intrabacterial "free" pool of cAMP.
Topics: Bacterial Proteins; Cyclic AMP; Heat-Shock Proteins; Microbial Viability; Mycobacterium smegmatis; Mycobacterium tuberculosis; Protein Binding
PubMed: 38636658
DOI: 10.1016/j.jbc.2024.107287 -
Journal of Proteomics May 2024Tuberculosis (TB) is a serious cause of infectious death worldwide. Recent studies have reported that about 30% of the Mtb proteome was modified post-translationally,...
Tuberculosis (TB) is a serious cause of infectious death worldwide. Recent studies have reported that about 30% of the Mtb proteome was modified post-translationally, indicating that their functions are essential for drug resistance, mycobacterial survival, and pathogenicity. Among them, lysine acetylation, reversibly regulated by acetyltransferase and deacetylase, has important roles involved in energy metabolism, cellular adaptation, and protein interactions. However, the substrate and biological functions of these two important regulatory enzymes remain unclear. Herein, we utilized the non-pathogenic M. smegmatis strain as a model and systematically investigated the dynamic proteome changes in response to the overexpressing of MsKat/MsCobB in mycobacteria. A total of 4179 proteins and 1236 acetylated sites were identified in our data. Further analysis of the dynamic changes involved in proteome and acetylome showed that MsKat/MsCobB played a regulatory role in various metabolic pathways and nucleic acid processes. After that, the quantitative mass spectrometric method was utilized and proved that the AMP-dependent synthetase, Citrate synthase, ATP-dependent specificity component of the Clp protease, and ATP-dependent DNA/RNA helicases were identified to be the substrates of MsKat. Overall, our study provided an important resource underlying the substrates and functions of the acetylation regulatory enzymes in mycobacteria. SIGNIFICANCE: In this study, we systematically analyzed the dynamic molecular changes in response to the MsKat/MsCobB overexpression in mycobacteria at proteome and lysine acetylation level by using a TMT-based quantitative proteomic approach. Pathways related with glycolysis, degradation of branched chain amino acids, phosphotransferase system were affected after disturbance of the two regulates enzymes involved in lysine acetylation. We also proved that AMP-dependent synthetase Clp protease, ATP-dependent DNA/RNA helicases and citrate synthase was the substrate of MsKat according to our proteomic data and biological validation. Together, our study underlined the substrates and functions of the acetylation regulatory enzymes in mycobacteria.
Topics: Mycobacterium smegmatis; Bacterial Proteins; Lysine Acetyltransferases; Acetylation; Proteome; Substrate Specificity; Lysine
PubMed: 38631426
DOI: 10.1016/j.jprot.2024.105177 -
Molecular Pharmaceutics May 2024Tuberculosis (TB) is a chronic disease caused by (Mtb), which shows a long treatment cycle often leads to drug resistance, making treatment more difficult. Immunogens...
Lipids Extracted from Mycobacterial Membrane and Enveloped PLGA Nanoparticles for Encapsulating Antibacterial Drugs Elicit Synergistic Antimicrobial Response against Mycobacteria.
Tuberculosis (TB) is a chronic disease caused by (Mtb), which shows a long treatment cycle often leads to drug resistance, making treatment more difficult. Immunogens present in the pathogen's cell membrane can stimulate endogenous immune responses. Therefore, an effective lipid-based vaccine or drug delivery vehicle formulated from the pathogen's cell membrane can improve treatment outcomes. Herein, we extracted and characterized lipids from, and the extracts contained lipids belonging to numerous lipid classes and compounds typically found associated with mycobacteria. The extracted lipids were used to formulate biomimetic lipid reconstituted nanoparticles (LrNs) and LrNs-coated poly(lactic--glycolic acid) nanoparticles (PLGA-LrNs). Physiochemical characterization and results of morphology suggested that PLGA-LrNs exhibited enhanced stability compared with LrNs. And both of these two types of nanoparticles inhibited the growth of . After loading different drugs, PLGA-LrNs containing berberine or coptisine strongly and synergistically prevented the growth of . Altogether, the bacterial membrane lipids we extracted with antibacterial activity can be used as nanocarrier coating for synergistic antibacterial treatment of ─an alternative model of Mtb, which is expected as a novel therapeutic system for TB treatment.
Topics: Polylactic Acid-Polyglycolic Acid Copolymer; Nanoparticles; Mycobacterium smegmatis; Lipids; Drug Synergism; Cell Membrane; Anti-Bacterial Agents; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Antitubercular Agents; Mycobacterium; Berberine; Drug Carriers; Tuberculosis
PubMed: 38622497
DOI: 10.1021/acs.molpharmaceut.3c01001 -
Apoptosis : An International Journal on... Apr 2024Mycobacterium tuberculosis (Mtb) genome possesses a unique family called Proline-Glutamate/Proline-Proline-Glutamate (PE/PPE) gene family, exclusive to pathogenic...
Mycobacterium tuberculosis (Mtb) genome possesses a unique family called Proline-Glutamate/Proline-Proline-Glutamate (PE/PPE) gene family, exclusive to pathogenic mycobacterium. Some of these proteins are known to play role in virulence and immune response modulation, but many are still uncharacterized. This study investigated the role of C-terminal region of Rv1039c (PPE15) in inducing mitochondrial perturbations and macrophage apoptosis. Our in-silico studies revealed the disordered, coiled, and hydrophobic C-terminal region in Rv1039c has similarity with C-terminal of mitochondria-targeting pro-apoptotic host proteins. Wild type Rv1039c and C-terminal deleted Rv1039c (Rv1039c-/-Cterm) recombinant proteins were purified and their M. smegmatis knock-in strains were constructed which were used for in-vitro experiments. Confocal microscopy showed localization of Rv1039c to mitochondria of PMA-differentiated THP1 macrophages; and reduced mitochondrial membrane depolarization and production of mitochondrial superoxides were observed in response to Rv1039c-/-Cterm in comparison to full-length Rv1039c. The C-terminal region of Rv1039c was found to activate caspases 3, 7 and 9 along with upregulated expression of pro-apoptotic genes like Bax and Bim. Rv1039c-/-Cterm also reduced the Cytochrome-C release from the mitochondria and the expression of AnnexinV/PI positive and TUNEL positive cells as compared to Rv1039c. Additionally, Rv1039c was observed to upregulate the TLR4-NF-κB-TNF-α signalling whereas the same was downregulated in response to Rv1039c-/-Cterm. These findings suggested that the C-terminal region of Rv1039c is a molecular mimic of pro-apoptotic host proteins which induce mitochondria-dependent macrophage apoptosis and evoke host immune response. These observations enhance our understanding about the role of PE/PPE proteins at host-pathogen interface.
PubMed: 38615303
DOI: 10.1007/s10495-024-01965-2 -
European Journal of Clinical... Jun 2024Rapid, reliable identification of mycobacteria from positive cultures is essential for patient management, particularly for the differential diagnosis of Mycobacterium...
PURPOSE
Rapid, reliable identification of mycobacteria from positive cultures is essential for patient management, particularly for the differential diagnosis of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) species. The aim of the present study was to evaluate a new "In-Vitro-Diagnostic"-certified PCR kit, FluoroType®-Mycobacteria VER 1.0 (Hain Lifescience GmbH) for NTM and MTBC identification from cultures.
METHODS
Mycobacteria identification isolated from positive cultures during routine practice at the Lyon university hospital mycobacteria laboratory obtained by hsp65 amplification/sequencing were compared retrospectively and prospectively to those obtained by and the FluoroType®-Mycobacteria VER 1.0 kit.
RESULTS
The overall agreement between hsp65 amplification/sequencing and the FluoroType®-Mycobacteria VER 1.0 kit was 88.4% (84/95); 91.2% (52/57) for the retrospective period and 84.2% (32/38) for the prospective period. There were 9 (9.5%) minor discrepancies (species in the FluoroType®-Mycobacteria VER 1.0 database and identified at genus level): 4 during the retrospective period, 5 during the prospective period; and 2 (2.1%) major discrepancies (species in the FluoroType®-Mycobacteria VER 1.0 database and identified incorrectly to species level): 1 during the retrospective period (M. kumamotonense identified as M. abscessus subsp massiliense by the kit) and 1 during the prospective period (M. chimaera identified as M. smegmatis by the kit). Including concordant results at genus level and minor discrepancies, 17.9% (17/95) of strains were identified as Mycobacterium sp. by the FluoroType®-Mycobacteria-VER 1.0 kit.
CONCLUSION
The good performance of the FluoroType®-Mycobacteria-VER 1.0 kit with few major discrepancies could enable its use for first-line identification of positive mycobacteria cultures. However, an alternative identification method at least for reference laboratories is needed owing to the non-negligible proportion of NTM strains were identified at genus level.
Topics: Humans; Retrospective Studies; Prospective Studies; Nontuberculous Mycobacteria; France; Bacterial Proteins; Mycobacterium; Polymerase Chain Reaction; Mycobacterium Infections, Nontuberculous; Chaperonin 60; Molecular Diagnostic Techniques; Sensitivity and Specificity
PubMed: 38607578
DOI: 10.1007/s10096-024-04825-8 -
Journal of Bacteriology May 2024In most actinomycetes, GlnR governs both nitrogen and non-nitrogen metabolisms (e.g., carbon, phosphate, and secondary metabolisms). Although GlnR has been recognized as...
UNLABELLED
In most actinomycetes, GlnR governs both nitrogen and non-nitrogen metabolisms (e.g., carbon, phosphate, and secondary metabolisms). Although GlnR has been recognized as a global regulator, its regulatory role in central carbon metabolism [e.g., glycolysis, gluconeogenesis, and the tricarboxylic acid (TCA) cycle] is largely unknown. In this study, we characterized GlnR as a direct transcriptional repressor of the gene that encodes phosphoenolpyruvate carboxykinase, catalyzing the conversion of the TCA cycle intermediate oxaloacetate to phosphoenolpyruvate, a key step in gluconeogenesis. Through the transcriptomic and quantitative real-time PCR analyses, we first showed that the transcription was upregulated in the null mutant of . Next, we proved that the gene was essential for gluconeogenesis when the TCA cycle intermediate was used as a sole carbon source. Furthermore, with the employment of the electrophoretic mobility shift assay and DNase I footprinting assay, we revealed that GlnR was able to specifically bind to the promoter region from both and two other representative actinomycetes ( and ). Therefore, our data suggest that GlnR may repress transcription in actinomycetes, which highlights the global regulatory role of GlnR in both nitrogen and central carbon metabolisms in response to environmental nutrient stresses.
IMPORTANCE
The GlnR regulator of actinomycetes controls nitrogen metabolism genes and many other genes involved in carbon, phosphate, and secondary metabolisms. Currently, the known GlnR-regulated genes in carbon metabolism are involved in the transport of carbon sources, the assimilation of short-chain fatty acid, and the 2-methylcitrate cycle, although little is known about the relationship between GlnR and the TCA cycle and gluconeogenesis. Here, based on the biochemical and genetic results, we identified GlnR as a direct transcriptional repressor of , the gene that encodes phosphoenolpyruvate carboxykinase, a key enzyme for gluconeogenesis, thus highlighting that GlnR plays a central and complex role for dynamic orchestration of cellular carbon, nitrogen, and phosphate fluxes and bioactive secondary metabolites in actinomycetes to adapt to changing surroundings.
Topics: Gene Expression Regulation, Bacterial; Gluconeogenesis; Bacterial Proteins; Nitrogen; Repressor Proteins; Amycolatopsis; Promoter Regions, Genetic; Phosphoenolpyruvate Carboxykinase (ATP); Citric Acid Cycle; Actinobacteria
PubMed: 38606980
DOI: 10.1128/jb.00003-24 -
Journal of Clinical Tuberculosis and... May 2024Antimicrobial resistance alongside other challenges in tuberculosis (TB) therapeutics have stirred renewed interest in host-directed interventions, including the role of...
Antimicrobial resistance alongside other challenges in tuberculosis (TB) therapeutics have stirred renewed interest in host-directed interventions, including the role of antibodies as adjunct therapeutic agents. This study assessed the binding efficacy of two novel IgG1 opsonic monoclonal antibodies (MABs; GG9 & JG7) at 5, 10, and 25 µg/mL to live cultures of , , , , and American Type Culture Collection laboratory reference strains, as well as clinical susceptible, multi-drug resistant, and extensively drug resistant strains using indirect enzyme-linked immunosorbent assays. These three MAB concentrations were selected from a range of concentrations used in previous optimization (binding and functional) assays. Both MABs bound to all mycobacterial species and sub-types tested, albeit to varying degrees. Statistically significant differences in MAB binding activity were observed when comparing the highest and lowest MAB concentrations (p < 0.05) for both MABs GG9 and JG7, irrespective of the resistance profile. Binding affinity increased with an increase in MAB concentration, and optimal binding was observed at 25 µg/mL. JG7 showed better binding activity than GG9. Both MABs also bound to five MOTT species, albeit at varied levels. This non-selective binding to different mycobacterial species suggests a potential role for GG9 and JG7 as adjunctive agents in anti-TB chemotherapy with the aim to enhance bacterial killing.
PubMed: 38601919
DOI: 10.1016/j.jctube.2024.100435 -
Heliyon Apr 2024A series of nine 2,3-disubstituted-quinazolin-4(3)-one derived Schiff bases and their three Cu(II) complexes was prepared and tested for their antimicrobial activities...
A series of nine 2,3-disubstituted-quinazolin-4(3)-one derived Schiff bases and their three Cu(II) complexes was prepared and tested for their antimicrobial activities against reference strains ATCC 29213 and ATCC 29212 and resistant clinical isolates of methicillin-resistant (MRSA) and vancomycin-resistant (VRE). All the substances were tested against HRa ATCC 25177, DSM 44162 and ATCC 700084. While anti-enterococcal and antimycobacterial activities were insignificant, 3-[()-(2-hydroxy-5-nitrobenzylidene)amino]-2-(2-hydroxy-5-nitrophenyl)-2,3-dihydroquinazolin-4(1)-one () and its Cu(II) complex () demonstrated bacteriostatic antistaphylococcal activity. In addition, both compounds, as well as the other two prepared complexes, showed antibiofilm activity, which resulted in a reduction of biofilm formation and eradication of mature biofilm by 80% even at concentrations lower than the values of their minimum inhibitory concentrations. In addition, the compounds were tested for their cytotoxic effect on the human monocytic leukemia cell line THP-1. The antileukemic efficiency was improved by the preparation of Cu(II) complexes from the corresponding non-chelated Schiff base ligands.
PubMed: 38601653
DOI: 10.1016/j.heliyon.2024.e29051 -
Nature Communications Apr 2024Transcriptional regulation is a critical adaptive mechanism that allows bacteria to respond to changing environments, yet the concept of transcriptional plasticity (TP)...
Transcriptional regulation is a critical adaptive mechanism that allows bacteria to respond to changing environments, yet the concept of transcriptional plasticity (TP) - the variability of gene expression in response to environmental changes - remains largely unexplored. In this study, we investigate the genome-wide TP profiles of Mycobacterium tuberculosis (Mtb) genes by analyzing 894 RNA sequencing samples derived from 73 different environmental conditions. Our data reveal that Mtb genes exhibit significant TP variation that correlates with gene function and gene essentiality. We also find that critical genetic features, such as gene length, GC content, and operon size independently impose constraints on TP, beyond trans-regulation. By extending our analysis to include two other Mycobacterium species -- M. smegmatis and M. abscessus -- we demonstrate a striking conservation of the TP landscape. This study provides a comprehensive understanding of the TP exhibited by mycobacteria genes, shedding light on this significant, yet understudied, genetic feature encoded in bacterial genomes.
Topics: Mycobacterium tuberculosis; Bacterial Proteins; Genome, Bacterial; Operon; Mycobacterium smegmatis; Gene Expression Regulation, Bacterial
PubMed: 38600064
DOI: 10.1038/s41467-024-47410-5 -
Plants (Basel, Switzerland) Mar 2024The formation of biofilms underscores the challenge of treating bacterial infections. The study aimed to assess the antioxidant, cytotoxicity, antibacterial,...
The formation of biofilms underscores the challenge of treating bacterial infections. The study aimed to assess the antioxidant, cytotoxicity, antibacterial, anti-motility, and anti-biofilm effects of defatted fractions from (resurrection plant). Antioxidant activity was assessed using DPPH radical scavenging and hydrogen peroxide assays. Cytotoxicity was screened using a brine shrimp lethality assay. Antibacterial activity was determined using the micro-dilution and growth curve assays. Antibiofilm potential was screened using the crystal violet and tetrazolium reduction assay. Liquid-liquid extraction of crude extracts concentrated polyphenols in the ethyl acetate and n-butanol fractions. Subsequently, these fractions had notable antioxidant activity and demonstrated broad-spectrum antibacterial activity against selected Gram-negative and Gram-positive bacteria and (MIC values < 630 μg/mL). Growth curves showed that the bacteriostatic inhibition by the ethyl acetate fractions was through the extension of the lag phase and/or suppression of the growth rate. The sub-inhibitory concentrations of the ethyl acetate fractions inhibited the swarming motility of and by 100% and eradicated more than 50% of biofilm biomass. The polyphenolic content of plays an important role in its antibacterial, anti-motility, and antibiofilm activity, thus offering an additional strategy to treat biofilm-associated infections.
PubMed: 38592866
DOI: 10.3390/plants13060847