Recombinant PGL/p-HBAD biosynthesis glycosyltransferase Rv2958c/MT3034 (Rv2958c, MT3034)

What is the genomic organization and basic characteristics of Rv2958c/MT3034?

Rv2958c (MT3034 in CDC1551 strain) is located on the negative strand of the M. tuberculosis genome at position 3310714-3312000. The gene is 1,287 base pairs in length and encodes a protein of 428-429 amino acids . It belongs to the UDP-glucoronosyl and UDP-glucosyltransferases family of proteins and functions as a glycosyltransferase involved in the biosynthesis of phenolic glycolipids (PGL) and p-hydroxybenzoic acid derivatives (p-HBAD) .

The basic characteristics of Rv2958c can be summarized in the following table:

What are the known functional roles of Rv2958c in M. tuberculosis biology?

Rv2958c plays several critical roles in M. tuberculosis biology, primarily centered around cell wall component synthesis and host-pathogen interactions:

• Glycolipid biosynthesis: Rv2958c functions as a glycosyltransferase involved in the biosynthesis of phenolic glycolipids (PGL) and p-hydroxybenzoic acid derivatives (p-HBAD) .

• Immune evasion: The protein is associated with evasion or tolerance of host immune responses, making it important for pathogenesis .

• Cholesterol metabolism: Experimental data indicates that this gene is required for growth on cholesterol, suggesting its involvement in lipid metabolism pathways that are critical during infection .

• Transferase activity: Specifically, Rv2958c demonstrates transferase activity by transferring hexosyl groups during the biosynthesis of cell wall components .

• Pathogenesis: The protein contributes to the pathogenic capabilities of M. tuberculosis, likely through its role in synthesizing cell wall components that interact with the host .

For investigating these functions experimentally, researchers typically employ gene knockout studies, complementation assays, and biochemical characterization of the purified recombinant protein.

How is Rv2958c regulated at the transcriptional level, and what co-regulated genes might function together with it?

Rv2958c exhibits specific patterns of co-regulation that provide insights into its functional networks:

Rv2958c is predicted to be co-regulated in two specific biclusters:

• Bicluster_0253 with a residual value of 0.45

• Bicluster_0479 with a residual value of 0.49

This regulation is potentially mediated by de-novo identified cis-regulatory motifs with the following e-values:

• Bicluster_0253: motifs with e-values of 18.00 and 8,100.00

• Bicluster_0479: motifs with e-values of 1,900.00 and 4,400.00

The co-regulated modules are enriched for genes associated with the following GO terms:

• Substrate-specific transmembrane transport

• Substrate-specific transporter activity

• Hydrogen ion transmembrane transporter activity

• Cation transmembrane transporter activity

• Transmembrane transporter activity

• Monovalent inorganic cation transmembrane transport

This co-regulation pattern suggests that Rv2958c functions within a network of genes involved in membrane transport processes, potentially coordinating cell wall component biosynthesis with transport mechanisms. To investigate this co-regulation experimentally, researchers could employ chromatin immunoprecipitation (ChIP) assays to identify transcription factors binding to the Rv2958c promoter region, or use electrophoretic mobility shift assays (EMSA) to confirm specific DNA-protein interactions.

What is the relationship between Rv2958c and other enzymes in the PGL and p-HBAD biosynthetic pathways?

Rv2958c functions as part of a complex biosynthetic network for PGL and p-HBAD production. The search results reveal important relationships with other enzymes in these pathways:

• Operon structure: Rv2959c is part of an operon that includes Rv2958c, indicating coordinated expression of these functionally related genes .

• Functional relationship with methyltransferases: Rv2958c works in conjunction with methyltransferases encoded by Rv2952 and Rv2959c. Specifically:

  • The protein encoded by Rv2952 catalyzes the transfer of a methyl group onto the lipid moiety of phthiotriol and glycosylated phenolphthiotriol dimycocerosates to form DIM and PGL .

  • The enzyme encoded by Rv2959c catalyzes the O-methylation of the hydroxyl group located on carbon 2 of the rhamnosyl residue linked to the phenolic group of PGL and p-HBAD .

• Sequential enzymatic actions: The biosynthesis of PGL and p-HBAD involves multiple enzymatic steps where Rv2958c catalyzes glycosyl transfer reactions that are followed or preceded by methylation steps catalyzed by the enzymes mentioned above .

To study these relationships experimentally, researchers should consider enzyme assays with purified recombinant proteins, metabolite profiling of knockout mutants, and protein-protein interaction studies using techniques such as bacterial two-hybrid systems or co-immunoprecipitation.

How do genetic variations in Rv2958c differ between M. tuberculosis strains and related mycobacterial species?

Analysis of genetic variations in Rv2958c across mycobacterial strains reveals important evolutionary insights:

• Conservation in M. tuberculosis strains: The Rv2958c gene is highly conserved between the two major sequenced strains of M. tuberculosis (H37Rv and CDC1551), showing no frameshift mutations .

• Variation in M. bovis: Interestingly, while many genes show identical frameshift patterns across M. tuberculosis and M. bovis strains, Rv2958c does not appear among the shared Insertion/Deletion Containing Sequences (ICDSs) listed in the comparative genomic studies .

• Functional implications: The absence of Rv2958c in the list of common ICDSs suggests that this gene has maintained its integrity throughout the evolution of pathogenic mycobacteria, highlighting its potential importance for the core functions of these organisms .

• Evolutionary significance: The conservation of intact Rv2958c across pathogenic mycobacterial species suggests selective pressure to maintain this gene's function, likely due to its role in pathogenesis and immune evasion .

For experimental investigation of these variations, researchers should consider comparative genomic analyses across a wider range of clinical isolates, phylogenetic analyses of Rv2958c sequences, and functional complementation studies using orthologs from different species.

What methodological approaches are most effective for expressing and purifying recombinant Rv2958c for enzymatic studies?

To effectively express and purify recombinant Rv2958c for enzymatic studies, researchers should consider the following methodological approaches:

• Expression systems:

  • E. coli-based expression: BL21(DE3) or Rosetta strains with pET vectors containing codon-optimized Rv2958c sequence can provide good expression levels.

  • Mycobacterial expression systems: Consider M. smegmatis expression systems for proper folding and post-translational modifications.

• Solubility enhancement strategies:

  • Fusion tags: MBP (maltose-binding protein) or SUMO tags can significantly enhance solubility.

  • Expression conditions: Lower temperatures (16-18°C) and reduced IPTG concentrations often improve soluble protein yield.

  • Co-expression with chaperones: GroEL/GroES system can assist with proper folding.

• Purification protocol:

  • Initial capture: Nickel or cobalt affinity chromatography with His-tagged constructs.

  • Intermediate purification: Ion exchange chromatography (IEX) based on the theoretical pI of Rv2958c.

  • Polishing: Size exclusion chromatography to remove aggregates and ensure homogeneity.

• Activity assessment:

  • Substrate spectrum analysis using UDP-glucose, UDP-galactose, and other potential UDP-activated sugars.

  • HPLC or mass spectrometry-based assays to detect glycosylation products.

  • Isothermal titration calorimetry (ITC) for binding studies with potential substrates.

While the search results don't provide specific purification protocols for Rv2958c, these approaches are based on standard methods for glycosyltransferases and can be optimized for this specific protein.

What is the role of Rv2958c in M. tuberculosis virulence and host-pathogen interactions?

Rv2958c contributes significantly to M. tuberculosis virulence and host-pathogen interactions through several mechanisms:

• Immune evasion: The gene ontology data indicates that Rv2958c is involved in "evasion or tolerance of host immune response," suggesting that its products help shield the bacterium from host defenses .

• Cell wall integrity: As a glycosyltransferase involved in PGL and p-HBAD biosynthesis, Rv2958c contributes to the unique cell wall structure of M. tuberculosis, which is known to be important for resistance to host defense mechanisms and antibiotics .

• Pathogenesis: Gene ontology data explicitly associates Rv2958c with pathogenesis, indicating its direct involvement in the disease-causing capabilities of M. tuberculosis .

• Virulence factor synthesis: The PGL and p-HBAD molecules synthesized by pathways involving Rv2958c have been shown to be "important virulence factors" in mycobacterial pathogenesis .

• Molecular camouflage: The glycosylated products generated through Rv2958c activity may contribute to molecular mimicry or camouflage strategies that help the bacterium avoid recognition by host immune cells .

Experimental approaches to investigate these roles include:

• Infection studies comparing wild-type and Rv2958c knockout strains

• Macrophage survival assays

• Cytokine profiling during infection with strains having modified Rv2958c

• Animal models evaluating disease progression and immune response

How can mutant strains of M. tuberculosis with altered Rv2958c function be generated and characterized?

Creating and characterizing Rv2958c mutants requires careful methodological approaches:

The search results indicate that mutants of Rv2958c are available, suggesting that these approaches have been successfully implemented .

What analytical techniques are most appropriate for studying the enzymatic activity of Rv2958c in vitro?

To effectively characterize the enzymatic activity of Rv2958c in vitro, researchers should consider several complementary analytical techniques:

• Radiochemical assays:

  • Using radiolabeled UDP-sugars (e.g., UDP-[14C]-glucose) to track the transfer of glycosyl groups to acceptor substrates.

  • Scintillation counting or autoradiography to quantify product formation.

• HPLC-based methods:

  • Reverse-phase HPLC to separate glycosylated and non-glycosylated forms of acceptor substrates.

  • HILIC (Hydrophilic Interaction Liquid Chromatography) for analyzing sugar nucleotides and reaction products.

• Mass spectrometry approaches:

  • MALDI-TOF or ESI-MS to determine the exact mass changes after glycosylation.

  • MS/MS fragmentation patterns to confirm glycosidic bond formation and positioning.

• Spectrophotometric assays:

  • Coupled enzyme assays that link glycosyl transfer to NAD+/NADH conversion for continuous monitoring.

  • Colorimetric detection of UDP release using specific reagents.

• Structural studies:

  • X-ray crystallography of Rv2958c alone and in complex with substrates.

  • Molecular docking simulations to predict substrate binding modes.

  • Site-directed mutagenesis of predicted catalytic residues followed by activity assays.

• Biophysical interaction analyses:

  • Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to measure binding affinities for different substrates.

  • Differential scanning fluorimetry (DSF) to assess thermal stability in the presence of substrates or inhibitors.

While the search results don't provide specific assay conditions for Rv2958c enzymatic activity, these techniques represent standard approaches in glycosyltransferase research and should be applicable to this enzyme.

How should researchers interpret discrepancies in experimental results when studying Rv2958c function across different M. tuberculosis strains?

When confronted with discrepancies in experimental results across different M. tuberculosis strains, researchers should consider several methodological approaches to interpretation:

• Genetic context analysis:

  • Examine the genomic environment of Rv2958c in each strain, as differences in adjacent genes or regulatory regions may affect function.

  • Verify whether Rv2958c is part of the same operon structure across strains, as noted in search result which identifies it in an operon with Rv2959c.

• Strain-specific variations:

  • Sequence the Rv2958c gene from each strain to identify potential single nucleotide polymorphisms or small indels not annotated in databases.

  • Consider the evolutionary relationships between strains, as detailed in search result , which describes phylogenetic links between M. tuberculosis strains.

• Experimental condition standardization:

  • Ensure identical growth conditions, as expression of Rv2958c may be influenced by factors like carbon source availability (particularly cholesterol) .

  • Standardize protein extraction and enzyme assay conditions to eliminate methodological variables.

• Complementation studies:

  • Express Rv2958c from one strain in another to determine if the observed differences are due to the gene itself or other strain-specific factors.

  • Use site-directed mutagenesis to convert sequence variations between strains and assess functional consequences.

• Integrate multiple data types:

  • Combine transcriptomic, proteomic, and metabolomic analyses to build a comprehensive picture of Rv2958c function in each strain.

  • Consider quantitative proteomics data, as mentioned in search result , to assess protein abundance differences.

By systematically addressing these aspects, researchers can determine whether discrepancies reflect true biological differences or are artifacts of experimental procedures.

What bioinformatic approaches can be used to predict substrate specificity and structural features of Rv2958c?

To predict substrate specificity and structural features of Rv2958c, researchers can employ several complementary bioinformatic approaches:

• Homology modeling and structural prediction:

  • Generate 3D structural models using templates from related glycosyltransferases with known crystal structures.

  • Refine models using molecular dynamics simulations to predict flexible regions and stable conformations.

  • Identify potential catalytic residues based on structural conservation across the UDP-glycosyltransferase family.

• Sequence-based analyses:

  • Multiple sequence alignment with characterized glycosyltransferases to identify conserved motifs associated with specific donor or acceptor preferences.

  • Analysis of UDP-glycosyltransferase signature domains and their variants in Rv2958c.

  • Conservation analysis across mycobacterial species to identify functionally important residues.

• Substrate binding site prediction:

  • Molecular docking simulations with various UDP-activated sugars to predict donor substrate preferences.

  • Binding pocket analysis to characterize size, charge distribution, and hydrophobicity patterns.

  • Virtual screening of potential acceptor molecules based on known p-HBAD and PGL intermediates.

• Functional residue prediction:

  • Identification of potential catalytic residues using tools that predict protein-ligand interaction sites.

  • Conservation analysis focused on residues in predicted binding pockets.

  • Integration with co-expression data to identify functional networks, as described in search result which mentions co-regulation in biclusters 0253 and 0479.

• Phylogenetic approaches:

  • Construction of phylogenetic trees with functionally characterized glycosyltransferases to infer potential substrates based on evolutionary relationships.

  • Comparative genomics across species with known p-HBAD and PGL production capabilities.

These approaches can guide experimental work by generating testable hypotheses about structure-function relationships in Rv2958c.

What are promising strategies for developing inhibitors targeting Rv2958c for potential therapeutic applications?

Developing effective inhibitors against Rv2958c requires systematic approaches spanning computational prediction to experimental validation:

• Structure-based drug design:

  • Generate high-quality structural models of Rv2958c through X-ray crystallography, cryo-EM, or advanced homology modeling.

  • Identify druggable pockets, particularly the active site where UDP-sugar binding occurs.

  • Conduct virtual screening campaigns targeting these sites with diverse chemical libraries.

• Mechanism-based inhibitor design:

  • Develop transition state analogs that mimic the reaction intermediate during glycosyl transfer.

  • Design donor substrate (UDP-sugar) analogs with modifications that prevent catalysis but maintain binding.

  • Target protein-protein interaction sites if Rv2958c functions in a complex with other biosynthetic enzymes.

• Fragment-based approaches:

  • Screen fragment libraries against Rv2958c using biophysical methods like DSF, NMR, or X-ray crystallography.

  • Develop fragment hits into lead compounds through medicinal chemistry optimization.

  • Link fragments binding to adjacent pockets to create high-affinity inhibitors.

• Natural product exploration:

  • Screen plant-derived compounds, particularly those known to target glycosyltransferases.

  • Investigate microbial secondary metabolites for Rv2958c inhibitory activity.

• Validation methodologies:

  • Develop robust in vitro enzyme assays for high-throughput screening.

  • Establish cell-based assays measuring PGL and p-HBAD production in M. tuberculosis.

  • Assess inhibitor specificity against human glycosyltransferases to minimize off-target effects.

• Therapeutic potential assessment:

  • Evaluate the effect of Rv2958c inhibition on M. tuberculosis virulence and immune evasion capabilities, given its role in these processes as indicated in search results and .

  • Test lead compounds in macrophage infection models and animal models of tuberculosis.

This approach leverages the established role of Rv2958c in virulence factor biosynthesis to develop potential novel therapeutics.

How might Rv2958c be utilized in developing new diagnostic tools for tuberculosis detection?

Rv2958c offers several promising avenues for developing novel tuberculosis diagnostic tools:

• Antibody-based detection systems:

  • Develop specific monoclonal antibodies against Rv2958c for use in ELISA or lateral flow assays.

  • Design immunoassays targeting either the protein itself or its glycolipid products (PGL, p-HBAD).

  • Implement multiplex detection systems combining Rv2958c with other M. tuberculosis biomarkers.

• Nucleic acid detection platforms:

  • Design PCR primers targeting Rv2958c for species-specific detection of M. tuberculosis.

  • Develop LAMP (Loop-mediated isothermal amplification) assays for point-of-care testing.

  • Create RNA-based detection systems targeting Rv2958c mRNA as markers of active infection.

• Glycolipid profiling:

  • Develop mass spectrometry-based methods to detect specific PGL and p-HBAD signatures in patient samples.

  • Create aptamer-based sensors for detecting specific glycolipid products of Rv2958c activity.

  • Implement chromatographic methods for glycolipid biomarker detection in minimally processed samples.

• Host response assessment:

  • Measure host antibody responses to Rv2958c or its products as indicators of infection.

  • Develop T-cell activation assays using Rv2958c-derived peptides.

  • Design biosensors detecting host inflammatory responses to Rv2958c-produced glycolipids.

• Integration with existing diagnostic platforms:

  • Supplement GeneXpert systems with Rv2958c-specific detection modules.

  • Combine with traditional methods like tuberculin skin tests for improved accuracy.

The specificities of these approaches are supported by search results indicating Rv2958c's role in producing compounds unique to pathogenic mycobacteria , which suggests potential for highly specific diagnostic applications.