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Cellular and Molecular Life Sciences :... May 2024Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de...
Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de novo synthesis and uptake of various amino acids from host cells for metabolic demands. Pyridoxal phosphate-dependent histidinol phosphate aminotransferase-HspAT enzyme is critically required for histidine biosynthesis. HspAT is involved in metabolic synthesis of histidine, phenylalanine, tyrosine, tryptophan, and novobiocin. We showed that M. tuberculosis Rv2231c is a conserved enzyme with HspAT activity. Rv2231c is a monomeric globular protein that contains α-helices and β-sheets. It is a secretory and cell wall-localized protein that regulates critical pathogenic attributes. Rv2231c enhances the survival and virulence of recombinant M. smegmatis in infected RAW264.7 macrophage cells. Rv2231c is recognized by the TLR4 innate immune receptor and modulates the host immune response by suppressing the secretion of the antibacterial pro-inflammatory cytokines TNF, IL-12, and IL-6. It also inhibits the expression of co-stimulatory molecules CD80 and CD86 along with antigen presenting molecule MHC-I on macrophage and suppresses reactive nitrogen species formation, thereby promoting M2 macrophage polarization. Recombinant M. smegmatis expressing Rv2231c inhibited apoptosis in macrophages, promoting efficient bacterial survival and proliferation, thereby increasing virulence. Our results indicate that Rv2231c is a moonlighting protein that regulates multiple functions of M. tuberculosis pathophysiology to increase its virulence. These mechanistic insights can be used to better understand the pathogenesis of M. tuberculosis and to design strategies for tuberculosis mitigation.
Topics: Mice; Mycobacterium tuberculosis; Animals; RAW 264.7 Cells; Virulence; Macrophages; Transaminases; Bacterial Proteins; Mycobacterium smegmatis; Cytokines; Toll-Like Receptor 4; Humans; Immunity, Innate; Host-Pathogen Interactions; Tuberculosis
PubMed: 38698289
DOI: 10.1007/s00018-024-05200-8 -
FEBS Letters May 2024Mycobacterium tuberculosis (M. tb) has a complex cell wall, composed largely of mycolic acids, that are crucial to its structural maintenance. The M. tb desaturase...
Mycobacterium tuberculosis (M. tb) has a complex cell wall, composed largely of mycolic acids, that are crucial to its structural maintenance. The M. tb desaturase A1 (DesA1) is an essential Ca-binding protein that catalyses a key step in mycolic acid biosynthesis. To investigate the structural and functional significance of Ca binding, we introduced mutations at key residues in its Ca-binding βγ-crystallin motif to generate DesA1F303A, E304Q, and F303A-E304Q. Complementation of a conditional ΔdesA1 strain of Mycobacterium smegmatis, with the Ca non-binders F303A or F303A-E304Q, failed to rescue its growth phenotype; these complements also exhibited enhanced cell wall permeability. Our findings highlight the criticality of Ca in DesA1 function, and its implicit role in the maintenance of mycobacterial cellular integrity.
PubMed: 38697952
DOI: 10.1002/1873-3468.14896 -
Journal of Biomolecular Structure &... Apr 2024tRNA-Encoded Peptides (tREPs), encoded by small open reading frames (smORFs) within tRNA genes, have recently emerged as a new class of functional peptides exhibiting...
tRNA-Encoded Peptides (tREPs), encoded by small open reading frames (smORFs) within tRNA genes, have recently emerged as a new class of functional peptides exhibiting antiparasitic activity. The discovery of tREPs has led to a re-evaluation of the role of tRNAs in biology and has expanded our understanding of the genetic code. This presents an immense, unexplored potential in the realm of tRNA-peptide interactions, paving the way for groundbreaking discoveries and innovative applications in various biological functions. This study explores the antimicrobial potential of tREPs against protein targets by employing a computational method that uses verified data sources and highly recognized predictive algorithms to provide a sorted list of likely antimicrobial peptides, which were then filtered for toxicity, cell permeability, allergenicity and half-life. These peptides were then docked with screened protein targets and computationally validated using molecular dynamics (MD) simulations for 150 ns and the binding free energy was estimated. The peptides Pep2 (VVLWRKPRVRKTG) and Pep6 (HRLRLRRRKPWW) exhibited good binding affinities of -110.5 +/- 2.5 and -129.0 +/- 3.9, respectively, with RMSD values of 0.4 and 0.25 nm against the fucose-binding lectin (7NEF) and the 30S ribosome of (5O5J) protein targets. The 7NEF-Pep2 and 5O5J-Pep6 complexes indicated higher negative binding free energies of -52.55 kcal/mol and -55.52 kcal/mol respectively, as calculated by Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA). Thus, the tREPs derived peptides designed as a part of this study, provide novel approaches for potential anti-bacterial therapeutic modalities.Communicated by Ramaswamy H. Sarma.
PubMed: 38676533
DOI: 10.1080/07391102.2024.2335555 -
Molecules (Basel, Switzerland) Apr 2024Two α-pyrone analogs were isolated from the endophytic fungus sp. CB10100, which is derived from the medicinal plant . These analogs included a new compound,...
Two α-pyrone analogs were isolated from the endophytic fungus sp. CB10100, which is derived from the medicinal plant . These analogs included a new compound, diaporpyrone F (), and a known compound, diaporpyrone D (). The structure of was identified by a comprehensive examination of HRESIMS, 1D and 2D NMR spectroscopic data. Bioinformatics analysis revealed that biosynthetic gene clusters for α-pyrone analogs are common in fungi of species. The in vitro α-glucosidase inhibitory activity and antibacterial assay of revealed that it has a 46.40% inhibitory effect on α-glucosidase at 800 μM, while no antibacterial activity against methicillin-resistant (MRSA), () or at 64 μg/mL. Molecular docking and molecular dynamics simulations of with α-glucosidase further suggested that the compounds are potential α-glucosidase inhibitors. Therefore, α-pyrone analogs can be used as lead compounds for α-glucosidase inhibitors in more in-depth studies.
Topics: Pyrones; Molecular Docking Simulation; Glycoside Hydrolase Inhibitors; Molecular Dynamics Simulation; alpha-Glucosidases; Ascomycota; Anti-Bacterial Agents; Molecular Structure; Microbial Sensitivity Tests
PubMed: 38675588
DOI: 10.3390/molecules29081768 -
Microorganisms Apr 2024Mycobacterial membrane proteins play a pivotal role in the bacterial invasion of host cells; however, the precise mechanisms underlying certain membrane proteins remain...
Mycobacterial membrane proteins play a pivotal role in the bacterial invasion of host cells; however, the precise mechanisms underlying certain membrane proteins remain elusive. (Ms) is a hemolysin III family protein that is homologous to (Mtb) , but it has an unclear function in growth. To address this issue, we utilized the CRISPR/Cas9 gene editor to construct Δ strains and combined RNA transcription and LC-MS/MS protein profiling to determine the functional role of in Ms growth. The correlative analysis showed that the deletion of inhibits ABC transporters in the cytomembrane and inhibits the biosynthesis of amino acids in the cell wall. Corresponding to these results, we confirmed that MSMEG5257 localizes in the cytomembrane via subcellular fractionation and also plays a role in facilitating the transport of iron ions in environments with low iron levels. Our data provide insights that plays a role in maintaining Ms metabolic homeostasis, and the deletion of significantly impacts the growth rate of Ms. Furthermore, , a promising drug target, offers a direction for the development of novel therapeutic strategies against mycobacterial diseases.
PubMed: 38674714
DOI: 10.3390/microorganisms12040770 -
Acta Crystallographica. Section F,... Apr 2024The rise in antimicrobial resistance is a global health crisis and necessitates the development of novel strategies to treat infections. For example, in 2022...
The rise in antimicrobial resistance is a global health crisis and necessitates the development of novel strategies to treat infections. For example, in 2022 tuberculosis (TB) was the second leading infectious killer after COVID-19, with multi-drug-resistant strains of TB having an ∼40% fatality rate. Targeting essential biosynthetic pathways in pathogens has proven to be successful for the development of novel antimicrobial treatments. Fatty-acid synthesis (FAS) in bacteria proceeds via the type II pathway, which is substantially different from the type I pathway utilized in animals. This makes bacterial fatty-acid biosynthesis (Fab) enzymes appealing as drug targets. FabG is an essential FASII enzyme, and some bacteria, such as Mycobacterium tuberculosis, the causative agent of TB, harbor multiple homologs. FabG4 is a conserved, high-molecular-weight FabG (HMwFabG) that was first identified in M. tuberculosis and is distinct from the canonical low-molecular-weight FabG. Here, structural and functional analyses of Mycolicibacterium smegmatis FabG4, the third HMwFabG studied to date, are reported. Crystal structures of NAD and apo MsFabG4, along with kinetic analyses, show that MsFabG4 preferentially binds and uses NADH when reducing CoA substrates. As M. smegmatis is often used as a model organism for M. tuberculosis, these studies may aid the development of drugs to treat TB and add to the growing body of research that distinguish HMwFabGs from the archetypal low-molecular-weight FabG.
Topics: Mycobacterium smegmatis; Bacterial Proteins; Crystallography, X-Ray; Models, Molecular; Amino Acid Sequence; Recombinant Proteins
PubMed: 38656226
DOI: 10.1107/S2053230X2400356X -
Current Opinion in Microbiology Jun 2024Members of the order Mycobacteriales are distinguished by a characteristic diderm cell envelope, setting them apart from other Actinobacteria species. In addition to the... (Review)
Review
Members of the order Mycobacteriales are distinguished by a characteristic diderm cell envelope, setting them apart from other Actinobacteria species. In addition to the conventional peptidoglycan cell wall, these organisms feature an extra polysaccharide polymer composed of arabinose and galactose, termed arabinogalactan. The nonreducing ends of arabinose are covalently linked to mycolic acids (MAs), forming the immobile inner leaflet of the highly hydrophobic MA membrane. The contiguous outer leaflet of the MA membrane comprises trehalose mycolates and various lipid species. Similar to all actinobacteria, Mycobacteriales exhibit apical growth, facilitated by a polar localized elongasome complex. A septal cell envelope synthesis machinery, the divisome, builds instead of the cell wall structures during cytokinesis. In recent years, a growing body of knowledge has emerged regarding the cell wall synthesizing complexes of Mycobacteriales., focusing particularly on three model species: Corynebacterium glutamicum, Mycobacterium smegmatis, and Mycobacterium tuberculosis.
Topics: Cell Wall; Mycolic Acids; Galactans; Peptidoglycan; Mycobacterium tuberculosis; Corynebacterium glutamicum; Mycobacterium smegmatis; Arabinose; Bacterial Proteins
PubMed: 38653035
DOI: 10.1016/j.mib.2024.102478 -
Microbial Pathogenesis Jun 2024Staphylococcus aureus is a major human pathogen that can cause infections that range from superficial skin and mucosal infections to life threatening disseminated...
Staphylococcus aureus is a major human pathogen that can cause infections that range from superficial skin and mucosal infections to life threatening disseminated infections. S. aureus can attach to medical devices and host tissues and form biofilms that allow the bacteria to evade the host immune system and provide protection from antimicrobial agents. To counter host-generated oxidative and nitrosative stress mechanisms that are part of the normal host responses to invading pathogens, S. aureus utilizes low molecular weight (LMW) thiols, such as bacillithiol (BSH). Additionally, S. aureus synthesizes its own nitric oxide (NO), which combined with its downstream metabolites may also protect the bacteria against specific host responses. We have previously shown that LMW thiols are required for biofilm formation in Mycobacterium smegmatis and Pseudomonas aeruginosa. Here, we show that the S. aureus bshC mutant strain, which is defective in the last step of the BSH pathway and lacks BSH, is impaired in biofilm formation. We also identify a possible S-nitrosobacillithiol reductase (BSNOR), similar in sequence to an S-nitrosomycothiol reductase found in M. smegmatis and show that the putative S. aureus bsnoR mutant strain has reduced levels of BSH and decreased biofilm formation. Our studies also show that NO plays an important role in biofilm formation and that acidified sodium nitrite severely reduces biofilm thickness. These studies provide insight into the roles of oxidative and nitrosative stress mechanisms on biofilm formation and indicate that BSH and NO are key players in normal biofilm formation in S. aureus.
Topics: Biofilms; Staphylococcus aureus; Glucosamine; Cysteine; Nitric Oxide; Sodium Nitrite; Bacterial Proteins; Mycobacterium smegmatis; Mutation; Humans; Oxidoreductases; Sulfhydryl Compounds; Oxidative Stress
PubMed: 38649100
DOI: 10.1016/j.micpath.2024.106657 -
Biochemistry. Biokhimiia Mar 2024The synthesis of (p)ppGpp alarmones plays a vital role in the regulation of metabolism suppression, growth rate control, virulence, bacterial persistence, and biofilm...
The synthesis of (p)ppGpp alarmones plays a vital role in the regulation of metabolism suppression, growth rate control, virulence, bacterial persistence, and biofilm formation. The (p)ppGpp alarmones are synthesized by proteins of the RelA/SpoT homolog (RSH) superfamily, including long bifunctional RSH proteins and small alarmone synthetases. Here, we investigated enzyme kinetics and dose-dependent enzyme inhibition to elucidate the mechanism of 4-(4,7-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)pentanoic acid (DMNP) action on the (p)ppGpp synthetases Rel and RelZ from Mycolicibacterium smegmatis and Rel from Mycobacterium tuberculosis. DMNP was found to inhibit the activity of Rel. According to the enzyme kinetics analysis, DMNP acts as a noncompetitive inhibitor of Rel and RelZ. Based on the results of molecular docking, the DMNP-binding site is located in the proximity of the synthetase domain active site. This study might help in the development of alarmone synthetase inhibitors, which includes relacin and its derivatives, as well as DMNP - a synthetic analog of the marine coral metabolite erogorgiaene. Unlike conventional antibiotics, alarmone synthetase inhibitors target metabolic pathways linked to the bacterial stringent response. Although these pathways are not essential for bacteria, they regulate the development of adaptation mechanisms. Combining conventional antibiotics that target actively growing cells with compounds that impede bacterial adaptation may address challenges associated with antimicrobial resistance and bacterial persistence.
Topics: Bacterial Proteins; Enzyme Inhibitors; Kinetics; Ligases; Molecular Docking Simulation; Mycobacterium smegmatis; Mycobacterium tuberculosis; Naphthalenes; Diterpenes
PubMed: 38648761
DOI: 10.1134/S0006297924030027 -
Scientific Reports Apr 2024Tuberculosis remains a large health threat, despite the availability of the tuberculosis vaccine, BCG. As BCG efficacy gradually decreases from adolescence, BCG-Prime...
Recombinant mycobacterial DNA-binding protein 1 with post-translational modifications boosts IFN-gamma production from BCG-vaccinated individuals' blood cells in combination with CpG-DNA.
Tuberculosis remains a large health threat, despite the availability of the tuberculosis vaccine, BCG. As BCG efficacy gradually decreases from adolescence, BCG-Prime and antigen-booster may be an efficient strategy to confer vaccine efficacy. Mycobacterial DNA-binding protein 1 (MDP1, namely Rv2986c, hupB or HU) is a major Mycobacterium tuberculosis protein that induces vaccine-efficacy by co-administration with CpG DNA. To produce MDP1 for booster-vaccine use, we have created recombinant MDP1 produced in both Escherichia coli (eMDP1) and Mycolicibacterium smegmatis (mMDP1), an avirulent rapid-growing mycobacteria. We tested their immunogenicity by checking interferon (IFN)-gamma production by stimulated peripheral blood cells derived from BCG-vaccinated individuals. Similar to native M. tuberculosis MDP1, we observed that most lysin resides in the C-terminal half of mMDP1 are highly methylated. In contrast, eMDP1 had less post-translational modifications and IFN-gamma stimulation. mMDP1 stimulated the highest amount of IFN-gamma production among the examined native M. tuberculosis proteins including immunodominant MPT32 and Antigen 85 complex. MDP1-mediated IFN-gamma production was more strongly enhanced when combined with a new type of CpG DNA G9.1 than any other tested CpG DNAs. Taken together, these results suggest that the combination of mMDP1 and G9.1 possess high potential use for human booster vaccine against tuberculosis.
Topics: Humans; Interferon-gamma; Bacterial Proteins; Protein Processing, Post-Translational; BCG Vaccine; DNA-Binding Proteins; Mycobacterium tuberculosis; Recombinant Proteins; Oligodeoxyribonucleotides; Tuberculosis; CpG Islands; Mycobacterium smegmatis; Escherichia coli; Female
PubMed: 38644371
DOI: 10.1038/s41598-024-58836-8