-
Nature Communications Mar 2024Natural herbs, which contain pharmacologically active compounds, have been used historically as medicines. Conventionally, the analysis of chemical components in herbal...
Natural herbs, which contain pharmacologically active compounds, have been used historically as medicines. Conventionally, the analysis of chemical components in herbal medicines requires time-consuming sample separation and state-of-the-art analytical instruments. Nanopore, a versatile single molecule sensor, might be suitable to identify bioactive compounds in natural herbs. Here, a phenylboronic acid appended Mycobacterium smegmatis porin A (MspA) nanopore is used as a sensor for herbal medicines. A variety of bioactive compounds based on salvianolic acids, including caffeic acid, protocatechuic acid, protocatechualdehyde, salvianic acid A, rosmarinic acid, lithospermic acid, salvianolic acid A and salvianolic acid B are identified. Using a custom machine learning algorithm, analyte identification is performed with an accuracy of 99.0%. This sensing principle is further used with natural herbs such as Salvia miltiorrhiza, Rosemary and Prunella vulgaris. No complex sample separation or purification is required and the sensing device is highly portable.
Topics: Nanopores; Plants, Medicinal; Algorithms; Plant Extracts; Alkenes; Polyphenols
PubMed: 38443335
DOI: 10.1038/s41467-024-45543-1 -
Biochemistry and Biophysics Reports Jul 2024Autophagy has emerged as a critical innate immune mechanism for host elimination of intracellular pathogens, however, the role of the autophagy receptor Optineurin...
Autophagy has emerged as a critical innate immune mechanism for host elimination of intracellular pathogens, however, the role of the autophagy receptor Optineurin during mycobacterial infection is not fully understood. To address this lacuna, we infected bone marrow-derived macrophages (BMDMs) derived from Optn and Optn mice with , and observed the infection outcome at sequential time points. While low multiplicity of infection (MOI) did not show any significant difference between BMDMs from the two groups, at high MOI Optn mice-derived BMDMs showed significantly lower colony forming unit counts, as well as lower cell counts at 12 h and 24 h post-infection. Quantification of cell numbers and nuclear morphologies at various time points post-infection indicated a markedly higher cell death in the Optineurin-deficient macrophages. Optineurin-deficient BMDMs showed significantly lower levels of the autophagosomal protein LC3-II upon infection, indicating a potential role for Optineurin in regulating autophagy during mycobacterial infection. Moreover, when stimulated by bacterial LPS, Optineurin deficient macrophages, showed altered levels of the inflammatory cytokine pro-IL-1β. These observations taken together suggest a novel regulatory role for Optineurin during mycobacterial infection. Its deficiency leads to an impairment in macrophage responses, directly impacting the pathophysiology of infection.
PubMed: 38434142
DOI: 10.1016/j.bbrep.2024.101672 -
Frontiers in Microbiology 2024The prevalence of bacterial persisters is related to their phenotypic diversity and is responsible for the relapse of chronic infections. Tolerance to antibiotic therapy...
The prevalence of bacterial persisters is related to their phenotypic diversity and is responsible for the relapse of chronic infections. Tolerance to antibiotic therapy is the hallmark of bacterial persistence. In this study, we have screened a transposon library of mc155 strain using antibiotic tolerance, survival in mouse macrophages, and biofilm-forming ability of the mutants. Out of 10 thousand clones screened, we selected ten mutants defective in all the three phenotypes. Six mutants showed significantly lower persister abundance under different stress conditions. Insertions in three genes belonging to the pathways of oxidative phosphorylation (), biotin metabolism (), and oxidative metabolism , a flavoprotein monooxygenase, significantly reduced the number of live cells, suggesting their role in pathways promoting long-term survival. Another group that displayed a moderate reduction in CFU included a glycosyltransferase, , a hydrogenase subunit, (), and a DNA binding protein, . The study has revealed potential candidates likely to facilitate the long-term survival of . The findings offer new targets to develop antibiotics against persisters. Further, investigating the corresponding genes in may provide valuable leads in improving the treatment of chronic and persistent tuberculosis infections.
PubMed: 38410395
DOI: 10.3389/fmicb.2024.1302883 -
MicroPublication Biology 2024Copper homeostasis plays a crucial role in mycobacteria. In , Rv0474 is a copper-responsive regulator with a copper-binding motif but its homolog in , MSMEG_0918,...
Copper homeostasis plays a crucial role in mycobacteria. In , Rv0474 is a copper-responsive regulator with a copper-binding motif but its homolog in , MSMEG_0918, lacks the copper-binding motif. We generated MSMEG_0918 knockdown strains of using CRISPRi. We confirmed the strains had varying levels of expression using RT-PCR. We demonstrated that MSMEG_0918 under-expression did not alter the growth of in standard aerobic culture as compared to the wild-type. Our knockdown strains (CHROME1 and CHROME2) could be further used towards understanding the role of MSMEG_0918 in .
PubMed: 38404920
DOI: 10.17912/micropub.biology.000891 -
Microbiology (Reading, England) Feb 2024Metal homeostasis is maintained by the uptake, storage and efflux of metal ions that are necessary for the survival of the bacterium. Homeostasis is mostly regulated by...
Metal homeostasis is maintained by the uptake, storage and efflux of metal ions that are necessary for the survival of the bacterium. Homeostasis is mostly regulated by a group of transporters categorized as ABC transporters and P-type ATPases. On the other hand, efflux pumps often play a role in drug-metal cross-resistance. Here, with the help of antibiotic sensitivity, antibiotic/dye accumulation and semi-quantitative biofilm formation assessments we report the ability of Rv3270, a P-type ATPase known for its role in combating Mn and Zn metal ion toxicity in , in influencing the extrusion of multiple structurally unrelated drugs and enhancing the biofilm formation of and Overexpression of Rv3270 increased the tolerance of host cells to norfloxacin, ofloxacin, sparfloxacin, ampicillin, oxacillin, amikacin and isoniazid. A significantly lower accumulation of norfloxacin, ethidium bromide, bocillin FL and levofloxacin in cells harbouring Rv3270 as compared to host cells indicated its role in enhancing efflux activity. Although over-expression of Rv3270 did not alter the susceptibility levels of levofloxacin, rifampicin and apramycin, the presence of a sub-inhibitory concentration of Zn resulted in low-level tolerance towards these drugs. Of note, the expression of Rv3270 enhanced the biofilm-forming ability of the host cells strengthening its role in antimicrobial resistance. Therefore, the study indicated that the over-expression of Rv3270 enhances the drug efflux activity of the micro-organism where zinc might facilitate drug-metal cross-resistance for some antibiotics.
Topics: Mycobacterium tuberculosis; P-type ATPases; Levofloxacin; Norfloxacin; Anti-Bacterial Agents; Oxacillin; Carrier Proteins
PubMed: 38373028
DOI: 10.1099/mic.0.001441 -
The Journal of Biological Chemistry Mar 2024In Mycobacterium smegmatis, the transcriptional activity of the alternative sigma factor SigF is posttranslationally regulated by the partner switching system consisting...
In Mycobacterium smegmatis, the transcriptional activity of the alternative sigma factor SigF is posttranslationally regulated by the partner switching system consisting of SigF, the anti-SigF RsbW1, and three anti-SigF antagonists (RsfA, RsfB, and RsbW3). We previously demonstrated that expression of the SigF regulon is strongly induced in the Δaa mutant of M. smegmatis lacking the aa cytochrome c oxidase, the major terminal oxidase in the respiratory electron transport chain. Here, we identified and characterized the RsfSR two-component system involved in regulating the phosphorylation state of the major anti-SigF antagonist RsfB. RsfS (MSMEG_6130) is a histidine kinase with the cyclase/histidine kinase-associated sensing extracellular 3 domain at its N terminus, and RsfR (MSMEG_6131) is a receiver domain-containing protein phosphatase 2C-type phosphatase that can dephosphorylate phosphorylated RsfB. We demonstrated that phosphorylation of RsfR on Asp74 by RsfS reduces the phosphatase activity of RsfR toward phosphorylated RsfB and that the cellular abundance of the active unphosphorylated RsfB is increased in the Δaa mutant relative to the WT strain. We also demonstrated that the RsfSR two-component system is required for induction of the SigF regulon under respiration-inhibitory conditions such as inactivation of the cytochrome bcc complex and aa cytochrome c oxidase, as well as hypoxia, electron donor-limiting, high ionic strength, and low pH conditions. Collectively, our results reveal a key regulatory element involved in regulating the SigF signaling system by monitoring the state of the respiratory electron transport chain.
Topics: Bacterial Proteins; Electron Transport; Electron Transport Complex IV; Gene Expression Regulation, Bacterial; Histidine Kinase; Mycobacterium smegmatis; Phosphoric Monoester Hydrolases; Sigma Factor
PubMed: 38367670
DOI: 10.1016/j.jbc.2024.105764 -
The Journal of Biological Chemistry Mar 2024Galactan polymer is a prominent component of the mycobacterial cell wall core. Its biogenesis starts at the cytoplasmic side of the plasma membrane by a build-up of the...
Galactan polymer is a prominent component of the mycobacterial cell wall core. Its biogenesis starts at the cytoplasmic side of the plasma membrane by a build-up of the linker disaccharide [rhamnosyl (Rha) - N-acetyl-glucosaminyl (GlcNAc) phosphate] on the decaprenyl-phosphate carrier. This decaprenyl-P-P-GlcNAc-Rha intermediate is extended by two bifunctional galactosyl transferases, GlfT1 and GlfT2, and then it is translocated to the periplasmic space by an ABC transporter Wzm-Wzt. The cell wall core synthesis is finalized by the action of an array of arabinosyl transferases, mycolyl transferases, and ligases that catalyze an attachment of the arabinogalactan polymer to peptidoglycan through the linker region. Based on visualization of the GlfT2 enzyme fused with fluorescent tags it was proposed that galactan polymerization takes place in a specific compartment of the mycobacterial cell envelope, the intracellular membrane domain, representing pure plasma membrane free of cell wall components (previously denoted as the "PMf" domain), which localizes to the polar region of mycobacteria. In this work, we examined the activity of the galactan-producing cellular machine in the cell-wall containing cell envelope fraction and in the cell wall-free plasma membrane fraction prepared from Mycobacterium smegmatis by the enzyme assays using radioactively labeled substrate UDP-[C]-galactose as a tracer. We found that despite a high abundance of GlfT2 in both of these fractions as confirmed by their thorough proteomic analyses, galactan is produced only in the reaction mixtures containing the cell wall components. Our findings open the discussion about the distribution of GlfT2 and the regulation of its activity in mycobacteria.
Topics: Galactans; Polymers; Proteomics; Transferases; Mycobacterium
PubMed: 38367664
DOI: 10.1016/j.jbc.2024.105768 -
Protein Science : a Publication of the... Mar 2024Outer membrane proteins perform essential functions in uptake and secretion processes in bacteria. MspA is an octameric channel protein in the outer membrane of...
Outer membrane proteins perform essential functions in uptake and secretion processes in bacteria. MspA is an octameric channel protein in the outer membrane of Mycobacterium smegmatis and is structurally distinct from any other known outer membrane protein. MspA is the founding member of a family with more than 3000 homologs and is one of the most widely used proteins in nanotechnological applications due to its advantageous pore structure and extraordinary stability. While a conserved C-terminal signal sequence is essential for folding and protein assembly in the outer membrane of Gram-negative bacteria, the molecular determinants of these processes are unknown for MspA. In this study, we show that mutation and deletion of methionine 183 in the highly conserved C-terminus of MspA and mutation of the conserved tryptophan 40 lead to a complete loss of protein in heat extracts of M. smegmatis. Swapping these residues partially restores the heat stability of MspA indicating that methionine 183 and tryptophan 40 form a conserved sulfur-π electron interaction, which stabilizes the MspA monomer. Flow cytometry showed that all MspA mutants are surface-accessible demonstrating that oligomerization and membrane integration in M. smegmatis are not affected. Thus, the conserved C-terminus of MspA is essential for its thermal stability, but it is not required for protein assembly in its native membrane, indicating that this process is mediated by a mechanism distinct from that in Gram-negative bacteria. These findings will benefit the rational design of MspA-like pores to tailor their properties in current and future applications.
Topics: Tryptophan; Porins; Mycobacterium; Mycobacterium smegmatis; Methionine
PubMed: 38358254
DOI: 10.1002/pro.4912 -
Nucleic Acids Research May 2024Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other...
Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other types of RNAs binding to RNAP exist in bacteria. To identify all RNAs interacting with RNAP and the primary σ factors, we have established and performed native RIP-seq in Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis. Besides known 6S RNAs in B. subtilis and Ms1 in M. smegmatis, we detected MTS2823, a homologue of Ms1, on RNAP in M. tuberculosis. In C. glutamicum, we discovered novel types of structured RNAs that associate with RNAP. Furthermore, we identified other species-specific RNAs including full-length mRNAs, revealing a previously unknown landscape of RNAs interacting with the bacterial transcription machinery.
Topics: Bacillus subtilis; Bacterial Proteins; Corynebacterium glutamicum; DNA-Directed RNA Polymerases; Gene Expression Regulation, Bacterial; Mycobacterium smegmatis; Mycobacterium tuberculosis; Nucleic Acid Conformation; RNA, Bacterial; RNA, Untranslated; Sigma Factor; Streptomyces coelicolor; Transcription, Genetic
PubMed: 38348908
DOI: 10.1093/nar/gkae081 -
Science Advances Feb 2024D29 mycobacteriophage encodes LysA endolysin, which mediates mycobacterial host cell lysis by targeting its peptidoglycan layer, thus projecting itself as a potential...
D29 mycobacteriophage encodes LysA endolysin, which mediates mycobacterial host cell lysis by targeting its peptidoglycan layer, thus projecting itself as a potential therapeutic. However, the regulatory mechanism of LysA during the phage lytic cycle remains ill defined. Here, we show that during D29 lytic cycle, structural and functional regulation of LysA not only orchestrates host cell lysis but also is critical for maintaining phage-host population dynamics by governing various phases of lytic cycle. We report that LysA exists in two conformations, of which only one is active, and the protein undergoes a host peptidoglycan-dependent conformational switch to become active for carrying out endogenous host cell lysis. D29 maintains a pool of inactive LysA, allowing complete assembly of phage progeny, thus helping avoid premature host lysis. In addition, we show that the switch reverses after lysis, thus preventing exogenous targeting of bystanders, which otherwise negatively affects phage propagation in the environment.
Topics: Mycobacteriophages; Bacteriophages; Mycobacterium smegmatis; Peptidoglycan; Endopeptidases
PubMed: 38335296
DOI: 10.1126/sciadv.adh9812