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Frontiers in Immunology 2021Although (Mtb) is an intracellular pathogen in phagocytic cells, the factors and mechanisms by which they invade and persist in host cells are still not well...
Although (Mtb) is an intracellular pathogen in phagocytic cells, the factors and mechanisms by which they invade and persist in host cells are still not well understood. Characterization of the bacterial proteins modulating macrophage function is essential for understanding tuberculosis pathogenesis and bacterial virulence. Here we investigated the pathogenic role of the Rv2145c protein in stimulating IL-10 production. We first found that recombinant Rv2145c stimulated bone marrow-derived macrophages (BMDMs) to secrete IL-10, IL-6 and TNF-α but not IL-12p70 and to increase the expression of surface molecules through the MAPK, NF-κB, and TLR4 pathways and enhanced STAT3 activation and the expression of IL-10 receptor in Mtb-infected BMDMs. Rv2145c significantly enhanced intracellular Mtb growth in BMDMs compared with that in untreated cells, which was abrogated by STAT3 inhibition and IL-10 receptor (IL-10R) blockade. Expression of Rv2145c in () led to STAT3-dependent IL-10 production and enhancement of intracellular growth in BMDMs. Furthermore, the clearance of Rv2145c-expressing in the lungs and spleens of mice was delayed, and these effects were abrogated by administration of anti-IL-10R antibodies. Finally, all mice infected with Rv2145c-expressing died, but those infected with the vector control strain did not. Our data suggest that Rv2145c plays a role in creating a favorable environment for bacterial survival by modulating host signals.
Topics: Animals; Bacterial Proteins; Interleukin-10; Macrophage Activation; Macrophages; Mice; Microbial Viability; Mycobacterium smegmatis; Mycobacterium tuberculosis; Receptors, Interleukin-10; Recombinant Proteins; STAT3 Transcription Factor; Signal Transduction; Toll-Like Receptor 4; Virulence
PubMed: 34017340
DOI: 10.3389/fimmu.2021.666293 -
DNA and Cell Biology May 2023Tuberculosis is an important chronic and often fatal infectious disease mainly caused by the bacterium (Mtb). Mtb is one of the most successful pathogens that harbors...
Tuberculosis is an important chronic and often fatal infectious disease mainly caused by the bacterium (Mtb). Mtb is one of the most successful pathogens that harbors several potential virulence factors not found in nonpathogenic mycobacteria. As the Mtb cell envelope is closely associated with its virulence and resistance, it is very important to understand the cell envelope for better treatment of causative pathogen. There is increasing evidence that Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins are the major effectors of virulence and persistence encoded in the Mtb H37Rv genome. However, the function of PE8 has not been explored to date. In this study, we heterologously expressed PE8 in nonpathogenic, fast-growing to investigate the interaction between PE8 and the host to determine its possible biological functions. We found that recombinant cells expressing PE8 were less susceptible to sodium dodecyl sulfate-induced surface stress compared with those expressing the empty vector, suggesting that PE8 may be involved in stress responses. In addition, macrophages infected with PE8-expressing produced obviously lower levels of the proinflammatory factor IL-1β, IL-6, and TNF-α and higher levels of the inhibitory factor IL-10. We further found that PE8 promoted survival within macrophages by inhibiting late apoptosis of macrophages. Collectively, selective targeting of the PE/PPE protein family offers an untapped opportunity to the development of more effective and safer drugs against Mtb infection.
Topics: Cytokines; Mycobacterium tuberculosis; Bacterial Proteins; Host-Pathogen Interactions; Mycobacterium smegmatis; Apoptosis
PubMed: 37074124
DOI: 10.1089/dna.2022.0316 -
Protein Science : a Publication of the... Mar 2022Accumulation of formaldehyde, a highly reactive molecule, in the cell is toxic, and requires detoxification for the organism's survival. Mycothiol-dependent formaldehyde...
Accumulation of formaldehyde, a highly reactive molecule, in the cell is toxic, and requires detoxification for the organism's survival. Mycothiol-dependent formaldehyde dehydrogenase or S-nitrosomycothiol reductase (MscR) from Mycobacterium smegmatis and Mycobacterium tuberculosis was previously known for detoxifying formaldehyde and protecting the cell against nitrosative stress. We here show that M. smegmatis MscR exhibits a mycothiol-independent formaldehyde dehydrogenase (FDH) activity in vitro. Presence of zinc in the reaction enhances MscR activity, thus making it a zinc-dependent FDH. Interestingly, MscR utilizes only formaldehyde and no other primary aldehydes as its substrate in vitro, and M. smegmatis lacking mscR (ΔmscR) shows sensitivity exclusively toward formaldehyde. Bioinformatics analysis of MscRs from various bacteria reveals 10 positionally conserved cysteines, whose importance in structural stability and biological activity is not yet investigated. To explore the significance of these cysteines, we generated MscR single Cys variants by systematically replacing each cysteine with serine. All of the Cys variants except C39S and C309S are unable to show a complete rescue of ΔmscR on formaldehyde, show a significant loss of enzymatic activity in vitro, pronounced structural alterations as probed by circular dichroism, and loss of homotetramerization on size exclusion chromatography. Our data thus reveal the importance of intact cysteines in the structural stability and biological activity of MscR, which is a dedicated FDH in M. smegmatis, and shows ~84% identity with M. tuberculosis MscR. We believe that this knowledge will further help in the development of FDH as a potential drug target against M. tuberculosis infections.
Topics: Aldehyde Oxidoreductases; Bacterial Proteins; Cysteine; Mycobacterium smegmatis; Mycobacterium tuberculosis
PubMed: 34904319
DOI: 10.1002/pro.4258 -
PloS One 2021Reduction of nitrate to nitrite in bacteria is an essential step in the nitrogen cycle, catalysed by a variety of nitrate reductase (NR) enzymes. The soil dweller,...
Reduction of nitrate to nitrite in bacteria is an essential step in the nitrogen cycle, catalysed by a variety of nitrate reductase (NR) enzymes. The soil dweller, Mycobacterium smegmatis is able to assimilate nitrate and herein we set out to confirm the genetic basis for this by probing NR activity in mutants defective for putative nitrate reductase (NR) encoding genes. In addition to the annotated narB and narGHJI, bioinformatics identified three other putative NR-encoding genes: MSMEG_4206, MSMEG_2237 and MSMEG_6816. To assess the relative contribution of each, the corresponding gene loci were deleted using two-step allelic replacement, individually and in combination. The resulting strains were tested for their ability to assimilate nitrate and reduce nitrate under aerobic and anaerobic conditions, using nitrate assimilation and modified Griess assays. We demonstrated that narB, narGHJI, MSMEG_2237 and MSMEG_6816 were individually dispensable for nitrate assimilation and for nitrate reductase activity under aerobic and anaerobic conditions. Only deletion of MSMEG_4206 resulted in significant reduction in nitrate assimilation under aerobic conditions. These data confirm that in M. smegmatis, narB, narGHJI, MSMEG_2237 and MSMEG_6816 are not required for nitrate reduction as MSMEG_4206 serves as the sole assimilatory NR.
Topics: Bacterial Proteins; Gene Deletion; Mycobacterium smegmatis; Nitrate Reductase; Nitrates
PubMed: 33471823
DOI: 10.1371/journal.pone.0245745 -
Journal of Interferon & Cytokine... Oct 2022The PE/PPE family proteins of have been associated with its virulence and interaction with the host immune system. The highly virulent modern lineage of possesses a...
The PE/PPE family proteins of have been associated with its virulence and interaction with the host immune system. The highly virulent modern lineage of possesses a lineage-specific PPE gene (), which arises from an ancestral mutation and is rarely studied. Here we examined the role of PPE7 in mycobacterial pathogenicity and survival by expressing PPE7 in . We show that, PPE7 activates host inflammation by increasing expression of pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6, while suppressing the expression of anti-inflammatory cytokines such as IL-10, possibly through the nuclear factor kappa B, ERK1/2, and p38 mitogen-activated protein kinase pathways. Overexpressing PPE7 in could enhance bacterial intracellular survival of infected macrophages. Furthermore, higher level of bacterial persistence, higher levels of TNF-α, IL-1β, and IL-6 cytokines, and more injury in the lung, liver, and spleen tissues of infected mice has been discovered. In conclusion, PPE7 could manipulate host immune response and increase bacterial persistence.
Topics: Mice; Animals; Mycobacterium smegmatis; Mycobacterium tuberculosis; NF-kappa B; Tumor Necrosis Factor-alpha; Interleukin-10; Interleukin-6; Mitogen-Activated Protein Kinases; Bacterial Proteins; Host-Pathogen Interactions; Cytokines; p38 Mitogen-Activated Protein Kinases; Tuberculosis
PubMed: 36178924
DOI: 10.1089/jir.2022.0062 -
Nucleic Acids Research Jul 2022Bacteria and bacteriophages co-evolve in a constant arms race, wherein one tries and finds newer ways to overcome the other. Phage resistance poses a great threat to the...
Bacteria and bacteriophages co-evolve in a constant arms race, wherein one tries and finds newer ways to overcome the other. Phage resistance poses a great threat to the development of phage therapy. Hence, it is both essential and important to understand the mechanism of phage resistance in bacteria. First identified in Mycobacterium smegmatis, the gene mpr, upon overexpression, confers resistance against D29 mycobacteriophage. Presently, the mechanism behind phage resistance by mpr is poorly understood. Here we show that Mpr is a membrane-bound DNA exonuclease, which digests DNA in a non-specific manner independent of the sequence, and shares no sequence or structural similarity with any known nuclease. Exonuclease activity of mpr provides resistance against phage infection, but the role of mpr may very well go beyond just phage resistance. Our experiments show that mpr plays a crucial role in the appearance of mutant colonies (phage resistant strains). However, the molecular mechanism behind the emergence of these mutant/resistant colonies is yet to be understood. Nevertheless, it appears that mpr is involved in the survival and evolution of M. smegmatis against phage. A similar mechanism may be present in other organisms, which requires further exploration.
Topics: Bacteriophages; Mycobacterium smegmatis
PubMed: 35713559
DOI: 10.1093/nar/gkac505 -
Journal of Clinical Tuberculosis and... Dec 2021Optimal management of infection with mycobacterial species requires accurate identification down to complex/species level due to variations in outcomes. Over the last...
Optimal management of infection with mycobacterial species requires accurate identification down to complex/species level due to variations in outcomes. Over the last few decades, there have been significant advances in laboratory diagnostics with development of newer and rapid molecular methods. Here we describe a case of that was misidentified as by DNA line probe assay.
PubMed: 34522793
DOI: 10.1016/j.jctube.2021.100268 -
ACS Chemical Biology May 2021In light of the continued threat of antimicrobial-resistant bacteria, new strategies to expand the repertoire of antimicrobial compounds are necessary. Prodrugs are an...
In light of the continued threat of antimicrobial-resistant bacteria, new strategies to expand the repertoire of antimicrobial compounds are necessary. Prodrugs are an underexploited strategy in this effort. Here, we report on the enhanced antimicrobial activity of a prodrug toward bacteria having an enzyme capable of its activation. A screen led us to the sulfurol ester of the antibiotic -3-(4-chlorobenzoyl)acrylic acid. An endogenous esterase makes sensitive to this prodrug. Candidate esterases were identified, and their heterologous production made sensitive to the ester prodrug. Taken together, these data suggest a new approach to the development of antimicrobial compounds that takes advantage of endogenous enzymatic activities to target specific bacteria.
Topics: Acrylates; Anti-Infective Agents; Bacillus subtilis; Drug Evaluation, Preclinical; Drug Resistance, Multiple, Bacterial; Escherichia coli; Esterases; Esters; Mycobacterium smegmatis; Prodrugs; Structure-Activity Relationship
PubMed: 33877811
DOI: 10.1021/acschembio.0c00894 -
Proceedings of the National Academy of... Aug 2020Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study...
Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study microbial ribosomal heterogeneity have mostly relied on comparing wild-type strains with mutants in which subunits have been deleted, but this approach does not allow direct comparison of alternate ribosome isoforms isolated from identical cellular contexts. Here, by simultaneously purifying canonical and alternative RpsR ribosomes from , we show that alternative ribosomes have distinct translational features compared with their canonical counterparts. Both alternative and canonical ribosomes actively take part in protein synthesis, although they translate a subset of genes with differential efficiency as measured by ribosome profiling. We also show that alternative ribosomes have a relative defect in initiation complex formation. Furthermore, a strain of in which the alternative ribosome protein operon is deleted grows poorly in iron-depleted medium, uncovering a role for alternative ribosomes in iron homeostasis. Our work confirms the distinct and nonredundant contribution of alternative bacterial ribosomes for adaptation to hostile environments.
Topics: Bacterial Proteins; Iron; Mycobacterium smegmatis; Peptide Chain Initiation, Translational; Protein Biosynthesis; Ribosomal Proteins; Ribosome Subunits; Ribosomes
PubMed: 32723820
DOI: 10.1073/pnas.2009607117 -
Frontiers in Cellular and Infection... 2023Mycobacteria assemble a complex cell wall with cross-linked peptidoglycan (PG) which plays an essential role in maintenance of cell wall integrity and tolerance to...
INTRODUCTION
Mycobacteria assemble a complex cell wall with cross-linked peptidoglycan (PG) which plays an essential role in maintenance of cell wall integrity and tolerance to osmotic pressure. We previously demonstrated that various hydrolytic enzymes are required to remodel PG during essential processes such as cell elongation and septal hydrolysis. Here, we explore the chemistry associated with PG cross-linking, specifically the requirement for amidation of the D-glutamate residue found in PG precursors.
METHODS
Synthetic fluorescent probes were used to assess PG remodelling dynamics in live bacteria. Fluorescence microscopy was used to assess protein localization in live bacteria and CRISPR-interference was used to construct targeted gene knockdown strains. Time-lapse microscopy was used to assess bacterial growth. Western blotting was used to assess protein phosphorylation.
RESULTS AND DISCUSSION
In , we confirmed the essentiality for D-glutamate amidation in PG biosynthesis by labelling cells with synthetic fluorescent PG probes carrying amidation modifications. We also used CRISPRi targeted knockdown of genes encoding the MurT-GatD complex, previously implicated in D-glutamate amidation, and demonstrated that these genes are essential for mycobacterial growth. We show that MurT-rseGFP co-localizes with mRFP-GatD at the cell poles and septum, which are the sites of cell wall synthesis in mycobacteria. Furthermore, time-lapse microscopic analysis of MurT-rseGFP localization, in fluorescent D-amino acid (FDAA)-labelled mycobacterial cells during growth, demonstrated co-localization with maturing PG, suggestive of a role for PG amidation during PG remodelling and repair. Depletion of MurT and GatD caused reduced PG cross-linking and increased sensitivity to lysozyme and β-lactam antibiotics. Cell growth inhibition was found to be the result of a shutdown of PG biosynthesis mediated by the serine/threonine protein kinase B (PknB) which senses uncross-linked PG. Collectively, these data demonstrate the essentiality of D-glutamate amidation in mycobacterial PG precursors and highlight the MurT-GatD complex as a novel drug target.
Topics: Amides; Glutamic Acid; Mycobacterium smegmatis; Cell Wall; Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; Bacterial Proteins; Peptidoglycan
PubMed: 37692163
DOI: 10.3389/fcimb.2023.1205829