Recombinant Uncharacterized protein Rv0048c/MT0054 (Rv0048c, MT0054)

What is Recombinant Uncharacterized protein Rv0048c/MT0054?

Recombinant Uncharacterized protein Rv0048c/MT0054 is a full-length protein encoded by the Rv0048c gene in Mycobacterium tuberculosis. The protein consists of 289 amino acids with the mature protein spanning residues 20-289. Current research utilizes recombinant versions of this protein with N-terminal His-tags expressed in E. coli expression systems for laboratory investigations . The protein remains functionally uncharacterized despite its potential significance in M. tuberculosis pathogenesis, particularly in the context of viable but non-replicating (VBNR) bacterial populations that may contribute to treatment failure or recurrence .

How is Rv0048c/MT0054 related to tuberculosis pathogenesis?

Rv0048c has been identified among a set of genes containing non-synonymous variants in clinical isolates of M. tuberculosis from patients with treatment failure or recurrence. Specifically, the V250A amino acid change has been observed in these clinical isolates, suggesting potential involvement in treatment outcomes . Research indicates that Rv0048c may be part of a genomic signature associated with M. tuberculosis strains that exhibit a higher propensity to form viable but non-replicating (VBNR) bacteria within infected macrophages. VBNR bacteria represent a subpopulation that maintains metabolic activity without replication, potentially contributing to persistence and treatment challenges in tuberculosis. Understanding the role of Rv0048c in this phenomenon requires methodological approaches involving dual fluorescence reporter systems to distinguish between actively replicating and non-replicating bacterial populations in experimental models .

What genetic variants of Rv0048c have been identified in clinical isolates?

The primary genetic variant of Rv0048c identified in clinical isolates is the V250A mutation, where a valine at position 250 is substituted with an alanine in the 289-amino acid protein . This variant has been specifically observed in M. tuberculosis isolates from patients with treatment failure or recurrence but was absent in isolates from successfully treated (cured) patients. The variant was identified through whole genome sequencing (WGS) of clinical isolates and comparative genomic analysis between treatment outcome groups. Researchers investigating similar variants should employ comparable methodologies, including culture of clinical isolates, DNA extraction, library preparation, WGS, and bioinformatic analysis of non-synonymous variants using appropriate reference genomes for alignment .

How does the V250A variant potentially affect Rv0048c function?

The V250A mutation represents a conservative substitution (both valine and alanine are hydrophobic amino acids, though alanine is smaller), which might subtly affect protein structure or function. While the exact functional impact remains uncharacterized, its association with treatment failure/recurrence suggests potential significance in bacterial survival or persistence . Methodologically, researchers can investigate the functional impact through comparative protein modeling, stability predictions, protein expression studies comparing wild-type and mutant proteins, and phenotypic characterization of recombinant M. tuberculosis strains carrying either variant. Additionally, site-directed mutagenesis experiments introducing this specific mutation into laboratory strains could help determine whether the variant directly contributes to persistence phenotypes or altered susceptibility to antitubercular agents.

What is the relationship between Rv0048c variants and VBNR M. tuberculosis formation?

Research has demonstrated that M. tuberculosis clinical isolates carrying the V250A variant in Rv0048c, along with other genetic variants, show an increased propensity to form viable but non-replicating (VBNR) bacteria during macrophage infection . VBNR bacteria maintain metabolic activity without undergoing replication, potentially contributing to treatment failure. The methodological approach to investigate this relationship involves:

• Dual fluorescence reporter systems utilizing constitutive GFP expression (measuring viability) and inducible red fluorescent protein (Turbo FP635) expression

• THP-1 macrophage infection models to recapitulate in vivo stressors

• Flow cytometric analysis to quantify high red fluorescence populations indicative of VBNR bacteria

• Comparative analysis between clinical isolates with and without the Rv0048c variant

This methodological framework provides a robust approach to investigating the functional significance of genetic variants in the context of bacterial persistence.

What are the optimal conditions for expressing and purifying Rv0048c/MT0054?

The optimal expression system for Rv0048c/MT0054 is E. coli with an N-terminal His-tag to facilitate purification . Researchers should consider the following methodological approach:

• Cloning the mature protein sequence (amino acids 20-289) into an appropriate expression vector with an N-terminal His-tag

• Expression in E. coli under standard induction conditions (typically IPTG induction)

• Purification via immobilized metal affinity chromatography (IMAC)

• Buffer optimization during purification to maintain protein stability

• Concentration determination via spectrophotometric methods or Bradford assay

• Lyophilization for long-term storage

Post-purification, the protein should be stored as a lyophilized powder and reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, the addition of 5-50% glycerol (final concentration) is recommended, with aliquots stored at -20°C/-80°C to avoid repeated freeze-thaw cycles, which can compromise protein integrity .

What analytical techniques are recommended for studying Rv0048c/MT0054 properties?

Multiple analytical techniques can be employed to characterize Rv0048c/MT0054:

• SDS-PAGE for purity assessment and molecular weight confirmation (>90% purity should be achieved)

• Circular dichroism (CD) spectroscopy to analyze secondary structure elements

• Size exclusion chromatography to determine oligomeric state

• Mass spectrometry for accurate molecular weight determination and post-translational modification analysis

• X-ray crystallography or cryo-EM for structural determination

• Computational structural predictions using homology modeling if experimental structures are unavailable

• Functional assays based on predicted biochemical activities

• Protein-protein interaction studies using pull-down assays, yeast two-hybrid, or co-immunoprecipitation

These methods should be applied systematically, starting with basic characterization (SDS-PAGE) and progressing to more complex structural and functional analyses to build a comprehensive understanding of this uncharacterized protein.

How can researchers investigate Rv0048c function in the context of M. tuberculosis persistence?

To investigate the potential role of Rv0048c in M. tuberculosis persistence, researchers should employ a multi-faceted approach:

• Macrophage infection model: Utilize THP-1 macrophages infected with M. tuberculosis strains expressing dual fluorescence reporters to distinguish between replicating and non-replicating bacteria

• Genetic manipulation: Generate knockout or knockdown strains of Rv0048c in laboratory M. tuberculosis strains

• Complementation studies: Reintroduce wild-type and V250A variant Rv0048c into knockout strains to assess functional restoration

• Stress response experiments: Evaluate the survival of Rv0048c-modified strains under various stressors including hypoxia, nutrient starvation, and antibiotic exposure

• Gene expression analysis: Compare transcriptional profiles of wild-type and Rv0048c-modified strains under standard and stress conditions

• Animal infection models: Assess the virulence and persistence of Rv0048c-modified strains in appropriate animal models

This comprehensive approach enables researchers to delineate the specific contribution of Rv0048c to bacterial persistence and potentially identify mechanisms by which the V250A variant might affect treatment outcomes.

What are the predicted structural features of Rv0048c/MT0054?

Based on amino acid sequence analysis, Rv0048c/MT0054 appears to have features consistent with a membrane-associated protein . The amino acid sequence (KDSDRQDACRILDDALRDGELSMEEHRERVSAATKAVTLGDLQRLVADLQVESAPAQMPALKSRAKRTELGLLAAAFVASVLLGVGIGWGVYGNTRSPLDFTSDPGAKPDGIAPVVLTPPRQLHSLGGLTGLLEQTRKRFGDTMGYRLVIYPEYASLDRVDPADDRRVLAYTYRGGWGDATSSAKSIADVSVVDLSKFDAKTAVGIMRGAPETLGLKQSDVKSMYLIVEPVKDPTTPAALSLSLYVSSDYGGGYLVFAGDGTIKHVSYPS) contains hydrophobic regions that may form transmembrane domains . To predict structural features, researchers should:

• Perform hydropathy analysis to identify potential membrane-spanning regions

• Use secondary structure prediction algorithms (e.g., PSIPRED, JPred)

• Apply protein fold recognition methods to identify structural homologs

• Utilize advanced structural prediction platforms like AlphaFold2 or RosettaFold

• Validate predictions through experimental approaches such as limited proteolysis, circular dichroism, or structural studies

These predictive analyses provide a foundation for hypothesis generation regarding protein function and guide subsequent experimental designs.

How might Rv0048c relate to other membrane proteins in M. tuberculosis?

The identification of Rv0048c variants in treatment-refractory clinical isolates, alongside numerous other membrane-associated proteins, suggests potential involvement in membrane-related functions that may contribute to bacterial persistence . Research indicates that 13 out of 23 shared non-synonymous variants in failed/recurrent TB isolates were in genes with membrane-related functions, including drug efflux proteins, secretion system components, and lipoproteins . To investigate the relationship between Rv0048c and other membrane proteins:

• Perform comparative genomic analysis across M. tuberculosis strains

• Conduct protein-protein interaction studies to identify binding partners

• Analyze co-expression patterns with known membrane proteins

• Examine physical clustering of genes encoding membrane proteins

• Investigate shared regulatory elements that may coordinate expression

This integrated approach would help position Rv0048c within the broader context of membrane-associated proteins that potentially contribute to M. tuberculosis virulence and persistence.

How can researchers differentiate between VBNR and dormant M. tuberculosis populations when studying Rv0048c?

Distinguishing viable but non-replicating (VBNR) bacteria from dormant populations is crucial for understanding persistence mechanisms potentially involving Rv0048c. The dual fluorescence reporter system described in the literature provides a methodological framework :

• Reporter system design: Use constitutive GFP expression to indicate cell viability and inducible red fluorescent protein (Turbo FP635) expression in the presence of an inducer (theophylline) to assess metabolic activity

• Quantification approach: Measure Turbo FP635 mean fluorescence intensities (MFI) at 0 hours and 120 hours post-infection

• Population identification: Define high red fluorescence populations based on MFI thresholds established at 0 hours

• Comparative analysis: Apply these gates to in vitro cultured bacteria and intracellular bacteria at later time points

• Flow cytometry applications: Utilize flow cytometry for quantitative assessment of population heterogeneity

This methodological approach enables researchers to specifically identify and quantify VBNR bacteria, distinguishing them from both actively replicating and completely dormant populations, providing insights into the potential role of Rv0048c in generating these subpopulations.

What genomic context should be considered when studying the role of Rv0048c in treatment failure?

Research indicates that Rv0048c is one of 23 genes with shared non-synonymous variants present in failed/recurrent TB isolates but absent in cured isolates . This suggests that Rv0048c should be studied within the broader genomic context rather than in isolation. Researchers should consider:

• The complete set of 23 shared variants (Table 4 from the research) :

• Enrichment of variants in membrane-associated functions (13/23 variants)

• Presence of variants in drug efflux proteins (Rv1218c, Stp)

• Variants in secretion system proteins (EspK) and ESAT-6-like proteins (EsxP)

• Variants in lipoproteins (LppA, LppB) with potential roles in virulence and immunoregulation

This holistic approach acknowledges that treatment failure likely results from combined effects of multiple genetic variants rather than Rv0048c alone, guiding more comprehensive experimental designs .

What computational approaches might help predict the function of Rv0048c?

Given the uncharacterized nature of Rv0048c, computational approaches offer valuable tools for function prediction:

• Homology-based methods: Identify distant homologs using position-specific scoring matrices and hidden Markov models (HMMs)

• Genomic context analysis: Examine gene neighborhood, gene fusion events, and co-occurrence patterns across bacterial genomes

• Protein-protein interaction predictions: Use interolog mapping and domain-based interaction predictions

• Structural insights: Apply template-based modeling and ab initio structure prediction methods like AlphaFold2

• Molecular dynamics simulations: Investigate potential binding sites and conformational flexibility

• Machine learning approaches: Train models on known protein functions to predict functions of uncharacterized proteins based on sequence features

• Phylogenetic profiling: Analyze patterns of gene presence/absence across diverse bacterial species

These computational strategies provide testable hypotheses about Rv0048c function that can guide experimental design and prioritization of biochemical assays.

How should researchers store and handle recombinant Rv0048c/MT0054 protein in laboratory settings?

Proper storage and handling of recombinant Rv0048c/MT0054 is critical for maintaining protein integrity and experimental reproducibility. Based on established protocols:

• Long-term storage: Maintain as lyophilized powder at -20°C/-80°C

• Reconstitution protocol: Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Working aliquots: Add glycerol to a final concentration of 5-50% (recommended default: 50%) before preparing aliquots for storage at -20°C/-80°C

• Working conditions: Store aliquots at 4°C for up to one week during active experimentation

• Freeze-thaw considerations: Avoid repeated freeze-thaw cycles that can compromise protein integrity

• Pre-use preparation: Briefly centrifuge vials before opening to bring contents to the bottom

Adherence to these storage and handling guidelines ensures protein stability and experimental consistency, particularly important when working with an uncharacterized protein where subtle functional characteristics may be crucial to discovery.

What quality control measures should be implemented when working with recombinant Rv0048c/MT0054?

Quality control is essential when working with recombinant proteins for research. For Rv0048c/MT0054, implement the following measures:

• Purity assessment: Confirm >90% purity via SDS-PAGE analysis

• Identity verification: Perform mass spectrometry analysis to confirm protein identity and molecular weight

• Functional integrity: Develop and apply functional assays appropriate to predicted activities

• Batch consistency: Establish lot-to-lot comparison protocols to ensure experimental reproducibility

• Contamination testing: Check for endotoxin contamination, particularly for proteins expressed in E. coli

• Storage validation: Periodically test stored aliquots to confirm retention of stability and activity

• Documentation: Maintain detailed records of expression conditions, purification methods, and quality control results

These measures ensure experimental reliability and facilitate troubleshooting if unexpected results occur during Rv0048c/MT0054 characterization studies.