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Nature Microbiology Apr 2023Mycobacteriophages are a diverse group of viruses infecting Mycobacterium with substantial therapeutic potential. However, as this potential becomes realized, the...
Mycobacteriophages are a diverse group of viruses infecting Mycobacterium with substantial therapeutic potential. However, as this potential becomes realized, the molecular details of phage infection and mechanisms of resistance remain ill-defined. Here we use live-cell fluorescence microscopy to visualize the spatiotemporal dynamics of mycobacteriophage infection in single cells and populations, showing that infection is dependent on the host nucleoid-associated Lsr2 protein. Mycobacteriophages preferentially adsorb at Mycobacterium smegmatis sites of new cell wall synthesis and following DNA injection, Lsr2 reorganizes away from host replication foci to establish zones of phage DNA replication (ZOPR). Cells lacking Lsr2 proceed through to cell lysis when infected but fail to generate consecutive phage bursts that trigger epidemic spread of phage particles to neighbouring cells. Many mycobacteriophages code for their own Lsr2-related proteins, and although their roles are unknown, they do not rescue the loss of host Lsr2.
Topics: Mycobacteriophages; Mycobacterium; Mycobacterium smegmatis; Bacteriophages
PubMed: 36823286
DOI: 10.1038/s41564-023-01333-x -
Journal of Bacteriology Jan 2020Stringent response is a conserved stress response mechanism in which bacteria employ the second messengers guanosine tetraphosphate and guanosine pentaphosphate...
Stringent response is a conserved stress response mechanism in which bacteria employ the second messengers guanosine tetraphosphate and guanosine pentaphosphate [collectively termed (p)ppGpp] to reprogram their cellular processes under stress. In mycobacteria, these alarmones govern a multitude of cellular phenotypes, such as cell division, biofilm formation, antibiotic tolerance, and long-term survival. possesses the bifunctional Rel as a (p)ppGpp synthetase and hydrolase. In addition, it contains a short alarmone synthetase MS_RHII-RSD (renamed RelZ), which contains an RNase H domain in tandem with the (p)ppGpp synthetase domain. The physiological functions of Rel have been well documented, but there is no clear picture about the cellular functions of RelZ in RelZ has been implicated in R-loop induced stress response due to its unique domain architecture. In this study, we elucidate the differential substrate utilization pattern of RelZ compared to that of Rel We unveil the ability of RelZ to use GMP as a substrate to synthesize pGpp, thereby expanding the repertoire of second messengers known in mycobacteria. We have demonstrated that the pGpp synthesis activity of RelZ is negatively regulated by RNA and pppGpp. Furthermore, we investigated its role in biofilm formation and antibiotic tolerance. Our findings highlight the complex role played by the RelZ in cellular physiology of and sheds light upon its functions distinct from those of Rel Bacteria utilize nucleotide messengers to survive the hostile environmental conditions and the onslaught of attacks within the host. The second messengers guanosine tetraphosphate and pentaphosphate [(p)ppGpp] have a profound impact on the long-term survival, biofilm formation, antibiotic tolerance, virulence, and pathogenesis of bacteria. Therefore, understanding the stress response mechanism regulated by (p)ppGpp is essential for discovering inhibitors of stress response and potential drug targets. contains two (p)ppGpp synthetases: Rel and RelZ. Our study unravels the novel regulatory mechanisms of RelZ activity and its role in mediating antibiotic tolerance. We further reveal its ability to synthesize novel second messenger pGpp, which may have regulatory roles in mycobacteria.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Gene Expression Regulation, Bacterial; Guanine Nucleotides; Guanosine Tetraphosphate; Mycobacterium smegmatis; Promoter Regions, Genetic
PubMed: 31659009
DOI: 10.1128/JB.00444-19 -
ELife Feb 2021Many antibiotics target the assembly of cell wall peptidoglycan, an essential, heteropolymeric mesh that encases most bacteria. In rod-shaped bacteria, cell wall...
Many antibiotics target the assembly of cell wall peptidoglycan, an essential, heteropolymeric mesh that encases most bacteria. In rod-shaped bacteria, cell wall elongation is spatially precise yet relies on limited pools of lipid-linked precursors that generate and are attracted to membrane disorder. By tracking enzymes, substrates, and products of peptidoglycan biosynthesis in , we show that precursors are made in plasma membrane domains that are laterally and biochemically distinct from sites of cell wall assembly. Membrane partitioning likely contributes to robust, orderly peptidoglycan synthesis, suggesting that these domains help template peptidoglycan synthesis. The cell wall-organizing protein DivIVA and the cell wall itself promote domain homeostasis. These data support a model in which the peptidoglycan polymer feeds back on its membrane template to maintain an environment conducive to directional synthesis. Our findings are applicable to rod-shaped bacteria that are phylogenetically distant from , indicating that horizontal compartmentalization of precursors may be a general feature of bacillary cell wall biogenesis.
Topics: Cell Cycle; Cell Membrane; Cell Wall; Mycobacterium smegmatis; Peptidoglycan
PubMed: 33544079
DOI: 10.7554/eLife.60263 -
Biochimica Et Biophysica Acta.... Jan 2022Tuberculosis (TB) remains a major cause of mortality and morbidity worldwide, and it is instant to discover novel anti-TB drugs due to the rapidly growing...
Tuberculosis (TB) remains a major cause of mortality and morbidity worldwide, and it is instant to discover novel anti-TB drugs due to the rapidly growing drug-resistance TB. Mycobacterium tuberculosis (Mtb) secreted effector ESAT6 plays a critical role in modulation miRNAs to regulate host defense mechanisms during Mtb infection, it can be a possible target for new tuberculosis drugs. The non-tuberculous mycobacteria Mycobacterium smegmatis (M. smegmatis) and Mtb have high gene homology but no pathogenicity. We used ESAT6 to interfere with macrophages or mice infected by M. smegmatis and determined that it enhanced the survival rate of bacteria and regulated miR-222-3p target PTEN. Expression of miR-222-3p reduced and PTEN enhanced with the progression of macrophages infected by M. smegmatis with ESAT6 co-incubation. MiR-222-3p overexpression diminished M. smegmatis survival and upregulated proinflammatory cytokines. VO-Ohpic trihydrate (PTEN inhibitor) reduced M. smegmatis survival and upregulated proinflammatory cytokines in vivo and in vitro, and VO-Ohpic trihydrate reversed the tissue damage of mouse organs caused by ESAT6. These results uncover an ESAT6 dependent role for miR-222-3p and its target PTEN in regulating host immune responses to bacterial infection and may provide a potential site for the development of anti-tuberculosis drugs that specifically antagonize the virulence of ESAT6.
Topics: Animals; Antigens, Bacterial; Bacterial Proteins; Disease Models, Animal; Host-Pathogen Interactions; Humans; Immunity, Innate; Mice; MicroRNAs; Mycobacterium smegmatis; Mycobacterium tuberculosis; PTEN Phosphohydrolase; Tuberculosis
PubMed: 34710568
DOI: 10.1016/j.bbadis.2021.166292 -
Microbiology (Reading, England) Feb 2022Virulent non-tuberculous Mycobacteria (NTMs) successfully reside and multiply within the phagosomes of phagocytic cells such as monocytes and macrophages. Macrophages...
Virulent non-tuberculous Mycobacteria (NTMs) successfully reside and multiply within the phagosomes of phagocytic cells such as monocytes and macrophages. Macrophages play a very important role in the innate clearance of intracellular pathogens including NTMs. Attenuated subsp. 100 enters macrophages but is incapable of escaping these cells via canonical mycobacteria escape mechanisms. Alternatively, virulent subsp. 104 and subsp. are able to modify macrophages to suit their growth, survival and ultimately escape from macrophages, while non-virulent is readily killed by macrophages. In this study we focused on early infection of macrophages with NTMs to determine the phenotypic response of macrophages, M1 or M2 differentiation, and phosphorylation alterations that can affect cellular response to invading bacteria. Our findings indicate that infection of the macrophage with MAH 100 and favours the development of M1 macrophage, a pro-inflammatory phenotype associated with the killing of intracellular pathogens, while infection of the macrophage with MAH 104 and favoured the development of M2 macrophage, an anti-inflammatory phenotype associated with the healing process. Interference with the host post-translational mechanisms, such as protein phosphorylation, is a key strategy used by many intracellular bacterial pathogens to modulate macrophage phenotype and subvert macrophage function. By comparing protein phosphorylation patterns of infected macrophages, we observed that uptake of both MAH 100 and resulted in MARCKS-related protein phosphorylation, which has been associated with macrophage activation. In contrast, in macrophages infected with MAH 104 and , methionine adenosyltransferase IIβ, an enzyme that catalyses the biosynthesis of S-adenosylmethionine, a methyl donor for DNA methylation. Inhibition of DNA methylation with 5-aza-2 deoxycytidine, significantly impaired the survival of MAH 104 in macrophages. Our findings suggest that the virulent MAH 104 and enhance its survival in the macrophage possibly through interference with the epigenome responses.
Topics: Macrophage Activation; Macrophages; Mycobacterium avium; Mycobacterium smegmatis
PubMed: 35133955
DOI: 10.1099/mic.0.001133 -
Acta Crystallographica. Section F,... Jul 2022The unintended crystallization of proteins which generally originate from the expression host instead of the target recombinant proteins is periodically reported....
The unintended crystallization of proteins which generally originate from the expression host instead of the target recombinant proteins is periodically reported. Despite the massive technological advances in the field, assigning a structural model to the corresponding diffraction data is not a trivial task. Here, the structure of acyl-carrier protein synthase (AcpS) from Mycobacterium smegmatis (msAcpS), which crystallized inadvertently in an experimental setup to grow crystals of a Mycobacterium tuberculosis protein using M. smegmatis as an expression system, is reported. After numerous unsuccessful attempts to solve the structure of the target protein by the molecular-replacement method no convincing solutions were obtained, indicating that the diffraction data may correspond to a crystal of an artifactual protein, which was finally identified by the Sequence-Independent Molecular replacement Based on Available Databases (SIMBAD) server. The msAcpS structure was solved at 2.27 Å resolution and structural analysis showed an overall conserved fold. msAcpS formed a trimeric structure similar to those of other reported structures of AcpS from various organisms; however, the residues involved in trimer formation are not strictly conserved. An unrelated metal ion (Ni), which was possibly incorporated during protein purification, was observed in the proximity of His49 and His116. Structural and sequence differences were observed in the loop connecting the α3 and α4 helices that is responsible for the open and closed conformations of the enzyme. Moreover, the structural analysis of msAcpS augments the current understanding of this enzyme, which plays a crucial role in the functional activation of acyl-carrier proteins in the fatty-acid biosynthesis pathway.
Topics: Acyl Carrier Protein; Bacterial Proteins; Crystallization; Crystallography, X-Ray; Mycobacterium smegmatis; Transferases
PubMed: 35787552
DOI: 10.1107/S2053230X22005738 -
MBio Mar 2021Functional characterization of bacterial proteins lags far behind the identification of new protein families. This is especially true for bacterial species that are more...
Functional characterization of bacterial proteins lags far behind the identification of new protein families. This is especially true for bacterial species that are more difficult to grow and genetically manipulate than model systems such as and To facilitate functional characterization of mycobacterial proteins, we have established a Mycobacterial Systems Resource (MSR) using the model organism This resource focuses specifically on 1,153 highly conserved core genes that are common to many mycobacterial species, including , in order to provide the most relevant information and resources for the mycobacterial research community. The MSR includes both biological and bioinformatic resources. The biological resource includes (i) an expression plasmid library of 1,116 genes fused to a fluorescent protein for determining protein localization; (ii) a library of 569 precise deletions of nonessential genes; and (iii) a set of 843 CRISPR-interference (CRISPRi) plasmids specifically targeted to silence expression of essential core genes and genes for which a precise deletion was not obtained. The bioinformatic resource includes information about individual genes and a detailed assessment of protein localization. We anticipate that integration of these initial functional analyses and the availability of the biological resource will facilitate studies of these core proteins in many species, including the less experimentally tractable pathogens , , , , , , and Diseases caused by mycobacterial species result in millions of deaths per year globally, and present a substantial health and economic burden, especially in immunocompromised patients. Difficulties inherent in working with mycobacterial pathogens have hampered the development and application of high-throughput genetics that can inform genome annotations and subsequent functional assays. To facilitate mycobacterial research, we have created a biological and bioinformatic resource (https://msrdb.org/) using as a model organism. The resource focuses specifically on 1,153 proteins that are highly conserved across the mycobacterial genus and, therefore, likely perform conserved mycobacterial core functions. Thus, functional insights from the MSR will apply to all mycobacterial species. We believe that the availability of this mycobacterial systems resource will accelerate research throughout the mycobacterial research community.
Topics: Computational Biology; Gene Library; Genes, Bacterial; Mycobacterium; Mycobacterium smegmatis; Research
PubMed: 33653882
DOI: 10.1128/mBio.02401-20 -
European Journal of Medicinal Chemistry Sep 2019Tuberculosis (TB) has recently become the leading killer among infectious diseases. Multidrug and extensively drug-resistant Mycobacterium tuberculosis strains urge the...
Tuberculosis (TB) has recently become the leading killer among infectious diseases. Multidrug and extensively drug-resistant Mycobacterium tuberculosis strains urge the need to develop anti-TB drugs with a novel mechanism of action. We describe synthesis of 22 novel imidazo[1,2-b][1,2,4,5]tetrazine derivatives with different substituents at C(3) and C(6) positions, and their antimycobacterial activity in vitro. 8 compounds show activity as potential serine/threonine protein kinase (STPK) inhibitors in M. smegmatis aphVIII+ test-system, which is characteristic for this class. 3 compounds out of 5 most active STPK inhibitors have a prominent minimal inhibitory concentration on M. tuberculosis H37Rv of 1 μg/ml. We were able to obtain M. smegmatis mc2 155 mutants resistant to 4 compounds and show that they do not have cross resistance with other drugs, but have a common mechanism of resistance among these 4 imidazo[1,2-b][1,2,4,5]tetrazines. Compound 3h seems the most promising, combining a predicted STPK inhibitor activity, the lowest MIC on M. tuberculosis and a low frequency of drug resistant mutants' emergence.
Topics: Antitubercular Agents; Drug Resistance, Microbial; Erythromycin; Heterocyclic Compounds, 2-Ring; Imidazoles; Imipenem; Microbial Sensitivity Tests; Molecular Structure; Mycobacterium smegmatis; Mycobacterium tuberculosis; Ofloxacin; Protein Serine-Threonine Kinases; Rifampin
PubMed: 31176094
DOI: 10.1016/j.ejmech.2019.05.081 -
Antibiotics (Basel, Switzerland) Sep 2021Antibiotic resistance is a growing problem and may become the next major global health crisis if no timely actions are taken. Mycobacterial infections are widespread...
Antibiotic resistance is a growing problem and may become the next major global health crisis if no timely actions are taken. Mycobacterial infections are widespread and, due to antibiotic resistance, also hard to treat and a major cause of mortality. Natural compounds have the potential to increase antibiotic effectiveness due to their resistance modulatory and antimicrobial effects. In this study, extracts, fractions, and isolated compounds were investigated regarding their antimicrobial and resistance-modulatory effects as well as efflux pump inhibition in extracts were found to have anti-mycobacterial potential and resistance modulating effects on ethidium bromide activity. The major antibacterial effect was attributed to ostruthin, and we found that the more lipophilic the substrate, the greater the antimicrobial effect. Imperatorin caused potent modulatory effects by interfering with the action of the major LfrA efflux pump in . The plant has a complex effect on including antibacterial, efflux pump inhibition, resistance modulation, and membrane permeabilization, and its major constituents, ostruthin and imperatorin, have a distinct role in these effects. This makes and its coumarins promising therapeutics to consider in the fight against drug-resistant mycobacteria.
PubMed: 34572657
DOI: 10.3390/antibiotics10091075 -
Acta Crystallographica. Section D,... Oct 2020Mycobacterium smegmatis MutT1 (MsMutT1) is a sanitation enzyme made up of an N-terminal Nudix hydrolase domain and a C-terminal domain resembling a histidine...
Mycobacterium smegmatis MutT1 (MsMutT1) is a sanitation enzyme made up of an N-terminal Nudix hydrolase domain and a C-terminal domain resembling a histidine phosphatase. It has been established that the action of MutT1 on 8-oxo-dGTP, 8-oxo-GTP and diadenosine polyphosphates is modulated by intermolecular interactions. In order to further explore this and to elucidate the structural basis of its differential action on 8-oxo-NTPs and unsubstituted NTPs, the crystal structures of complexes of MsMutT1 with 8-oxo-dGTP, GMPPNP and GMPPCP have been determined. Replacement soaking was used in order to ensure that the complexes were isomorphous to one another. Analysis of the structural data led to the elucidation of a relationship between the arrangements of molecules observed in the crystals, molecular plasticity and the action of the enzyme on nucleotides. The dominant mode of arrangement involving a head-to-tail sequence predominantly leads to the generation of NDPs. The other mode of packing arrangement appears to preferentially generate NMPs. This work also provides interesting insights into the dependence of enzyme action on the conformation of the ligand. The possibility of modulating the enzyme action through differences in intermolecular interactions and ligand conformations makes MsMutT1 a versatile enzyme.
Topics: Bacterial Proteins; Crystallography, X-Ray; Deoxyguanine Nucleotides; Ligands; Models, Molecular; Mycobacterium smegmatis; Protein Domains; Pyrophosphatases; Substrate Specificity; Nudix Hydrolases
PubMed: 33021500
DOI: 10.1107/S2059798320010992