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Hypoxanthine-Guanine Phosphoribosyltransferase Is Dispensable for Mycobacterium smegmatis Viability.Journal of Bacteriology Feb 2020Purine metabolism plays a ubiquitous role in the physiology of and other mycobacteria. The purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT)...
Purine metabolism plays a ubiquitous role in the physiology of and other mycobacteria. The purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is essential for growth ; however, its precise role in physiology is unclear. Membrane-permeable prodrugs of specifically designed HGPRT inhibitors arrest the growth of and represent potential new antituberculosis compounds. Here, we investigated the purine salvage pathway in the model organism Using genomic deletion analysis, we confirmed that HGPRT is the only guanine and hypoxanthine salvage enzyme in but is not required for growth of this mycobacterium or survival under long-term stationary-phase conditions. We also found that prodrugs of HGPRT inhibitors displayed an unexpected antimicrobial activity against that is independent of HGPRT. Our data point to a different mode of mechanism of action for these inhibitors than was originally proposed. Purine bases, released by the hydrolytic and phosphorolytic degradation of nucleic acids and nucleotides, can be salvaged and recycled. The hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which catalyzes the formation of guanosine-5'-monophosphate from guanine and inosine-5'-monophosphate from hypoxanthine, represents a potential target for specific inhibitor development. Deletion of the HGPRT gene () in the model organism confirmed that this enzyme is not essential for growth. Prodrugs of acyclic nucleoside phosphonates (ANPs), originally designed against HGPRT from , displayed anti- activities comparable to those obtained for but also inhibited the strain. These results confirmed that ANPs act in by a mechanism independent of HGPRT.
Topics: Antitubercular Agents; Catalysis; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hypoxanthine Phosphoribosyltransferase; Metabolic Networks and Pathways; Microbial Viability; Mycobacterium smegmatis; Plasmids; Purines
PubMed: 31818925
DOI: 10.1128/JB.00710-19 -
International Journal of Nanomedicine 2020Lipid polymer hybrid nanoparticles (LPHNPs) have been widely investigated in drug and gene delivery as well as in medical imaging. A knowledge of lipid-based surface...
BACKGROUND
Lipid polymer hybrid nanoparticles (LPHNPs) have been widely investigated in drug and gene delivery as well as in medical imaging. A knowledge of lipid-based surface engineering and its effects on how the physicochemical properties of LPHNPs affect the cell-nanoparticle interactions, and consequently how it influences the cytological response, is in high demand.
METHODS
Herein, we have engineered antibiotic-loaded (doxycycline or vancomycin) LPHNPs with cationic and zwitterionic lipids and examined the effects on their physicochemical characteristics (size and charge), antibiotic entrapment efficiency, and the in vitro intracellular bacterial killing efficiency against or infected macrophages.
RESULTS
The incorporation of cationic or zwitterionic lipids in the LPHNP formulation resulted in a size reduction in LPHNPs formulations and shifted the surface charge of bare NPs towards positive or neutral values. Also observed were influences on the drug incorporation efficiency and modulation of the drug release from the biodegradable polymeric core. The therapeutic efficacy of LPHNPs loaded with vancomycin was improved as its minimum inhibitory concentration (MIC) (2 µg/mL) versus free vancomycin (4 µg/mL). Importantly, our results show a direct relationship between the cationic surface nature of LPHNPs and its intracellular bacterial killing efficiency as the cationic doxycycline or vancomycin loaded LPHNPs reduced 4 or 3 log CFU respectively versus the untreated controls.
CONCLUSION
In our study, modulation of surface charge in the nanomaterial formulation increased macrophage uptake and intracellular bacterial killing efficiency of LPHNPs loaded with antibiotics, suggesting alternate way for optimizing their use in biomedical applications.
Topics: Animals; Anti-Bacterial Agents; Cell Line; Cell Survival; Drug Delivery Systems; Drug Liberation; Intracellular Space; Lipids; Macrophages; Mice; Microbial Sensitivity Tests; Mycobacterium smegmatis; Nanoparticles; Particle Size; Polymers; Staphylococcus aureus; Vancomycin
PubMed: 33162754
DOI: 10.2147/IJN.S271850 -
FEBS Letters May 2023Transcriptional factors such as the TetR family of transcriptional regulators (TFTRs) are widely found amongst bacteria, including mycobacteria, and are accountable for...
Transcriptional factors such as the TetR family of transcriptional regulators (TFTRs) are widely found amongst bacteria, including mycobacteria, and are accountable for their survival. Here, we characterized a novel TFTR, Ms6244, from Mycobacterium smegmatis that negatively autoregulates its expression and represses its neighbouring gene, Ms6243. We also report the binding of Ms6244 to the inverted repeats in the intergenic region of Ms6244 and Ms6243. Further, an Ms6244-deleted strain shows various morpho-physiological differences compared to the wild type. We further confirmed that the deletion of Ms6244 itself and not the resultant Ms6243 overexpression is the cause of the altered physiology. Our data thus suggest that Ms6244 is an essential regulator, having far-reaching effects on M. smegmatis physiology.
Topics: Mycobacterium smegmatis; Transcription Factors; Mycobacterium; Bacterial Proteins; Gene Expression Regulation, Bacterial
PubMed: 36694284
DOI: 10.1002/1873-3468.14582 -
Journal of Visualized Experiments : JoVE Dec 2023Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, including metabolites,...
Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, including metabolites, lipids, proteins, and nucleic acids, several groups have evaluated either the native or recombinant versions of bEVs for their protective potency as subunit vaccine candidates. Unlike native EVs, recombinant EVs are molecularly engineered to contain one or more immunogens of interest. Over the last decade, different groups have explored diverse approaches for generating recombinant bEVs. However, here, we report the design, construction, and enrichment of recombinant mycobacterial EVs (mEVs) in mycobacteria. Towards that, we use Mycobacterium smegmatis (Msm), an avirulent soil mycobacterium as the model system. We first describe the generation and enrichment of native EVs of Msm. Then, we describe the design and construction of recombinant mEVs that contain either mCherry, a red fluorescent reporter protein, or EsxA (Esat-6), a prominent immunogen of Mycobacterium tuberculosis. We achieve this by separately fusing mCherry and EsxA N-termini with the C-terminus of a small Msm protein Cfp-29. Cfp-29 is one of the few abundantly present proteins of MsmEVs. The protocol to generate and enrich recombinant mEVs from Msm remains identical to the generation and enrichment of native EVs of Msm.
Topics: Mycobacterium tuberculosis; Mycobacterium smegmatis; Extracellular Vesicles; Bacterial Proteins
PubMed: 38145372
DOI: 10.3791/65138 -
FASEB Journal : Official Publication of... Jul 2023Cholesterol is a crucial component in Mycobacterium tuberculosis virulence as it is required for phagocytosis of mycobacteria by macrophages. In addition, the tubercle...
Cholesterol is a crucial component in Mycobacterium tuberculosis virulence as it is required for phagocytosis of mycobacteria by macrophages. In addition, the tubercle bacilli can grow using cholesterol as the sole carbon source. Thus, cholesterol catabolism represents a valuable target for the development of new antitubercular drugs. However, the molecular partners of cholesterol catabolism remain elusive in mycobacteria. Here, we focused on HsaC and HsaD, enzymes involved in two consecutive steps of cholesterol ring degradation and identified putative partners, using a BirA-based proximity-dependent biotin identification (BioID) approach in Mycobacterium smegmatis. In rich medium, the fusion protein BirA-HsaD was able to fish the endogenous cognate HsaC, thus validating this approach to study protein-protein interactions and to infer metabolic channeling of cholesterol ring degradation. In chemically defined medium, both HsaC and HsaD interacted with four proteins, BkdA, BkdB, BkdC, and MSMEG_1634. BkdA, BkdB, and BkdC are enzymes that participate in the degradation of branched-chain amino acids. As cholesterol and branched-chain amino acid catabolism both generate propionyl-CoA, which is a toxic metabolite for mycobacteria, this interconnection suggests a compartmentalization to avoid dissemination of propionyl-CoA into the mycobacterial cytosol. Moreover, the BioID approach allowed us to decipher the interactome of MSMEG_1634 and MSMEG_6518, two proteins of unknown function, which are proximal to the enzymes involved in cholesterol and branched-chain amino acid catabolism. In conclusion, BioID is a powerful tool to characterize protein-protein interactions and to decipher the interconnections between different metabolic pathways, thereby facilitating the identification of new mycobacterial targets.
Topics: Animals; Mycobacterium smegmatis; Biotin; Cholesterol; Mycobacterium tuberculosis; Amino Acids, Branched-Chain; Bacterial Proteins
PubMed: 37331005
DOI: 10.1096/fj.202202018RR -
Methods in Molecular Biology (Clifton,... 2020Horizontal gene transfer (HGT) in prokaryotes disseminates genetic information throughout a population and can facilitate adaptation and evolution of the species....
Horizontal gene transfer (HGT) in prokaryotes disseminates genetic information throughout a population and can facilitate adaptation and evolution of the species. Mycobacteria utilize an atypical method of conjugation called distributive conjugal transfer (DCT), which results in mosaic genomes and the potential for accelerated evolution beyond that enabled by the more classical oriT-mediated conjugation. The following is a description of the basic DCT protocol, some possible variations of the assay, and examples of downstream applications to better understand mycobacterial functions.
Topics: Bacterial Physiological Phenomena; Conjugation, Genetic; DNA, Bacterial; Evolution, Molecular; Gene Transfer, Horizontal; Genome, Bacterial; High-Throughput Screening Assays; Mycobacterium smegmatis
PubMed: 31584159
DOI: 10.1007/978-1-4939-9877-7_9 -
Archives of Microbiology Dec 2021Tuberculosis (TB) causes millions of deaths each year across the globe. Multiple drug-resistant (MDR) and extensively drug-resistant (XDR) mycobacterial strains have...
Tuberculosis (TB) causes millions of deaths each year across the globe. Multiple drug-resistant (MDR) and extensively drug-resistant (XDR) mycobacterial strains have made the treatment extremely difficult. To overcome this hurdle, the development of new drug targets and an effective treatment strategy are desperately needed. This can be achieved by deciphering the role of essential genes and enzymes which are involved in cell survival. One such enzyme is glyoxalase II. The glyoxalase system (glyoxalase I and glyoxalase II) has a pivotal role in cellular survival and detoxification by converting methylglyoxal (MG) into lactate. Otherwise, the increased concentration of MG then modifies DNA, proteins, and lipids, resulting in abnormalities and cell death. Interestingly, the function and physiological role of glyoxalase II have remained undetermined in mycobacteria. In this study, the functional activity of MSMEG_2975 (putative glyoxalase II) after heterologous cloning and expression was determined. And the knockdown strain Mycobacterium smegmatis KD for MSMEG_2975 was constructed with tetracycline-inducible vector pMIND. The inducible knockdown of MSMEG_2975 affected bacterial growth, biofilm formation, transcriptome, and enhanced the susceptibility to antibiotics. This work represents mycobacterial glyoxalase II as a potential drug target against mycobacterial pathogens and indicates the crucial regulatory role of glyoxalase II in mycobacteria.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Mycobacterium smegmatis; Thiolester Hydrolases; Transcriptome
PubMed: 34964907
DOI: 10.1007/s00203-021-02652-5 -
Antimicrobial Agents and Chemotherapy Mar 2021Protein turnover via the Pup-proteasome system (PPS) is essential for nitric oxide resistance and virulence of , the causative agent of tuberculosis. Our study revealed...
Protein turnover via the Pup-proteasome system (PPS) is essential for nitric oxide resistance and virulence of , the causative agent of tuberculosis. Our study revealed components of PPS as novel determinants of intrinsic antifolate resistance in both and nonpathogenic The lack of expression of the prokaryotic ubiquitin-like protein (Pup) or the ligase, PafA, responsible for ligating Pup to its protein targets, enhanced antifolate susceptibility in Cross-species expression of homologs restored wild-type resistance to proteasomal mutants. Targeted deletion of and , encoding the structural components of the PPS proteolytic core, similarly resulted in reduced antifolate resistance. Furthermore, sulfonamides were synergistic with acidified nitrite, and the synergy against mycobacteria was enhanced in the absence of proteasomal activity. In , targeted mutagenesis followed by genetic complementation of , encoding the regulatory subunit responsible for translocating pupylated proteins to the proteolytic core, demonstrated a similar function of PPS in antifolate resistance. The overexpression of dihydrofolate reductase, responsible for the reduction of dihydrofolate to tetrahydrofolate, or disruption of the Lonely Guy gene, responsible for PPS-controlled production of cytokinins, abolished PPS-mediated antifolate sensitivity. Together, our results show that PPS protects mycobacteria from antimicrobial antifolates via regulating both folate reduction and cytokinin production.
Topics: Anti-Infective Agents; Bacterial Proteins; Folic Acid Antagonists; Mycobacterium smegmatis; Mycobacterium tuberculosis; Proteasome Endopeptidase Complex
PubMed: 33468462
DOI: 10.1128/AAC.01967-20 -
Journal of Microbiology (Seoul, Korea) Sep 2022Using a mutant of Mycobacterium smegmatis lacking the major aa cytochrome c oxidase of the electron transport chain (Δaa), we demonstrated that inhibition of the...
Activation of the SigE-SigB signaling pathway by inhibition of the respiratory electron transport chain and its effect on rifampicin resistance in Mycobacterium smegmatis.
Using a mutant of Mycobacterium smegmatis lacking the major aa cytochrome c oxidase of the electron transport chain (Δaa), we demonstrated that inhibition of the respiratory electron transport chain led to an increase in antibiotic resistance of M. smegmatis to isoniazid, rifampicin, ethambutol, and tetracycline. The alternative sigma factors SigB and SigE were shown to be involved in an increase in rifampicin resistance of M. smegmatis induced under respiration-inhibitory conditions. As in Mycobacterium tuberculosis, SigE and SigB form a hierarchical regulatory pathway in M. smegmatis through SigE-dependent transcription of sigB. Expression of sigB and sigE was demonstrated to increase in the Δaa mutant, leading to upregulation of the SigB-dependent genes in the mutant. The pho U2 (MSMEG_1605) gene implicated in a phosphate-signaling pathway and the MSMEG_1097 gene encoding a putative glycosyltransferase were identified to be involved in the SigB-dependent enhancement of rifampicin resistance observed for the Δaa mutant of M. smegmatis. The significance of this study is that the direct link between the functionality of the respiratory electron transport chain and antibiotic resistance in mycobacteria was demonstrated for the first time using an electron transport chain mutant rather than inhibitors of electron transport chain.
Topics: Bacterial Proteins; Electron Transport; Gene Expression Regulation, Bacterial; Mycobacterium smegmatis; Mycobacterium tuberculosis; Rifampin; Signal Transduction
PubMed: 35913593
DOI: 10.1007/s12275-022-2202-0 -
BMC Microbiology May 2022Bacteria require specialized secretion systems for the export of molecules into the extracellular space to modify their environment and scavenge for nutrients. The ESX-3...
BACKGROUND
Bacteria require specialized secretion systems for the export of molecules into the extracellular space to modify their environment and scavenge for nutrients. The ESX-3 secretion system is required by mycobacteria for iron homeostasis. The ESX-3 operon encodes for one cytoplasmic component (EccA) and five membrane components (EccB3 - EccE3 and MycP). In this study we sought to identify the sub-cellular location of EccA of the ESX-3 secretion system in mycobacteria.
RESULTS
Fluorescently tagged EccA localized to a single pole in the majority of Mycobacterium smegmatis cells and time-lapse fluorescent microscopy identified this pole as the growing pole. Deletion of ESX-3 did not prevent polar localization of fluorescently tagged EccA, suggesting that EccA unipolar localization is independent of other ESX-3 components. Affinity purification - mass spectrometry was used to identify EccA associated proteins which may contribute to the localization of EccA at the growing pole. EccA co-purified with fatty acid metabolism proteins (FAS, FadA3, KasA and KasB), mycolic acid synthesis proteins (UmaA, CmaA1), cell division proteins (FtsE and FtsZ), and cell shape and cell cycle proteins (MurS, CwsA and Wag31). Secretion system related proteins Ffh, SecA1, EccA1, and EspI were also identified.
CONCLUSIONS
Time-lapse microscopy demonstrated that EccA3 is located at the growing pole in M. smegmatis. The co-purification of EccA with proteins known to be required for polar growth, mycolic acid synthesis, the Sec secretion system (SecA1), and the signal recognition particle pathway (Ffh) also suggests that EccA is located at the site of active cell growth.
Topics: Bacterial Proteins; Mycobacterium; Mycobacterium smegmatis; Mycobacterium tuberculosis; Mycolic Acids; Operon
PubMed: 35590245
DOI: 10.1186/s12866-022-02554-6