Recombinant Nocardia farcinica Histidine--tRNA ligase (hisS)

What is Histidine--tRNA ligase (hisS) and what is its role in Nocardia farcinica metabolism?

Histidine--tRNA ligase (hisS) belongs to the aminoacyl-tRNA synthetase family and catalyzes the attachment of histidine to its cognate tRNA molecule (tRNAHis) in a two-step reaction. This enzyme first activates histidine using ATP to form histidyl-adenylate, followed by the transfer of the histidyl group to the 3' end of tRNAHis. In N. farcinica, hisS plays a critical role in protein synthesis by ensuring the correct incorporation of histidine into growing polypeptide chains.

The enzyme is essential for bacterial viability as protein synthesis cannot proceed correctly without functional aminoacyl-tRNA synthetases. While not directly identified as a virulence factor like Nfa34810, hisS is indispensable for the synthesis of all proteins in N. farcinica, including those involved in pathogenicity and survival within host cells . The bacterial hisS differs structurally from its human counterpart, making it a potential target for selective antimicrobial development.

How does N. farcinica infection manifestation inform research priorities for recombinant hisS studies?

N. farcinica infections can manifest as localized or disseminated disease, with the latter carrying a particularly high mortality rate. Studies have documented pneumonia with sepsis, multiple abscesses affecting the brain, lungs, and other tissues, and invasive disease even in immunocompetent hosts . This diverse clinical presentation highlights the importance of understanding N. farcinica's pathogenic mechanisms.

Research priorities for recombinant hisS studies should focus on:

• Investigating potential connections between protein synthesis capacity and virulence

• Exploring hisS as a potential biomarker for diagnostic applications (especially given the challenges in identifying Nocardia species)

• Developing hisS inhibitors as potential therapeutic agents against multidrug-resistant strains

• Understanding whether metabolic adaptations mediated by tRNA charging efficiency contribute to N. farcinica's ability to survive in diverse host environments

Researchers should particularly note that N. farcinica's ability to cause disseminated disease correlates with its invasive capacity, which depends on numerous virulence factors that require functional protein synthesis machinery .

What distinguishes research approaches for N. farcinica hisS from other bacterial aminoacyl-tRNA synthetases?

Research approaches for N. farcinica hisS require special considerations that distinguish them from studies of other bacterial aminoacyl-tRNA synthetases:

• Biosafety considerations: N. farcinica is a BSL-2 pathogen requiring appropriate containment facilities for work with native strains. Recombinant systems mitigate this concern but researchers must still validate that recombinant versions accurately represent native enzyme properties.

• Slow growth characteristics: Unlike model organisms like E. coli, N. farcinica has slower growth rates (≥3 days for colony formation), necessitating longer experimental timelines and specialized media for optimal expression .

• Cell wall complexity: N. farcinica possesses a complex cell wall containing mycolic acids, which impacts protein extraction protocols and may necessitate modified lysis procedures for native protein studies compared to Gram-negative models.

• Pathogenicity context: Research should consider hisS in the context of N. farcinica's documented ability to activate various immune pathways, including MAPK and NF-κB signaling cascades, as these may represent physiologically relevant environments for enzyme function .

Researchers approaching N. farcinica hisS studies should incorporate these distinctions into experimental design to ensure relevance to the organism's natural physiological context.

What expression systems yield optimal results for recombinant N. farcinica hisS production?

The selection of an expression system for recombinant N. farcinica hisS should balance protein yield, solubility, and retention of native enzymatic properties. Based on research experience with similar actinomycete proteins, the following expression systems demonstrate particular advantages:

For initial studies, an E. coli Rosetta(DE3) system with the pET28a(+) vector incorporating an N-terminal His6-tag typically provides the best compromise between yield and activity. Codon optimization is recommended as N. farcinica has a high G+C content (approximately 70%), which can lead to translational pausing in E. coli without rare codon supplementation.

To minimize inclusion body formation while maintaining reasonable yields, induction protocols should employ reduced temperature (18°C), moderate inducer concentration (0.2-0.3 mM IPTG), and extended expression time (16-20 hours). These conditions help emulate the slower protein synthesis rates characteristic of the native organism.

How can researchers optimize purification strategies for maximal enzymatic activity of recombinant N. farcinica hisS?

Purification of recombinant N. farcinica hisS requires careful attention to buffer composition and handling conditions to preserve enzymatic activity. A systematic approach involves:

• Initial capture: IMAC (Immobilized Metal Affinity Chromatography) using Ni-NTA resin with a buffer containing 50 mM HEPES (pH 7.5), 300 mM NaCl, 5% glycerol, and 2 mM β-mercaptoethanol. Imidazole should be used in a stepwise gradient (20-300 mM) to minimize non-specific binding while maximizing target protein recovery.

• Secondary purification: Size exclusion chromatography using Superdex 200 column in a buffer containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, and 1 mM DTT.

• Buffer optimization: Enzyme activity is typically highest in buffers containing 2-5 mM MgCl₂, which is essential for the ATP-dependent first step of the aminoacylation reaction.

Researchers should note that N. farcinica hisS, like other aminoacyl-tRNA synthetases, is sensitive to oxidation. Throughout purification, maintaining reducing conditions is crucial for preserving enzymatic activity. When studying immunogenic properties, it's important to remember that proteins from N. farcinica can elicit strong immune responses, as demonstrated with the Nfa34810 protein, which is immunodominant and recognized by sera from infected animals .

What analytical methods effectively verify the structural integrity and activity of purified recombinant N. farcinica hisS?

Comprehensive quality assessment of purified recombinant N. farcinica hisS requires multiple analytical approaches:

• Purity assessment:

  • SDS-PAGE (>95% purity expected)

  • Western blot with anti-His tag antibodies

  • Size exclusion chromatography-multi-angle light scattering (SEC-MALS) to confirm monodispersity and molecular weight

• Structural integrity verification:

  • Circular dichroism (CD) spectroscopy to assess secondary structure content

  • Thermal shift assay (TSA) to determine protein stability

  • Limited proteolysis to confirm proper folding

• Functional assessment:

  • ATP-PPi exchange assay to measure amino acid activation

  • Aminoacylation assay using [³H]-labeled histidine and purified tRNAHis

  • Enzyme kinetics determination (typical parameters shown below)

For researchers interested in immunological studies, it may be valuable to assess whether recombinant hisS is recognized by sera from patients or animals infected with N. farcinica, similar to the approach used with Nfa34810 . This could provide insights into whether hisS is expressed during infection and might serve as a diagnostic biomarker.

What structural approaches reveal crucial insights about N. farcinica hisS compared to human histidyl-tRNA synthetase?

Comparative structural analysis between N. farcinica hisS and human histidyl-tRNA synthetase provides crucial insights for both fundamental understanding and potential therapeutic development. Multiple approaches yield complementary information:

Key structural differences likely to be observed between N. farcinica and human histidyl-tRNA synthetases include:

• Active site architecture differences, particularly in the histidine binding pocket

• Differences in the ATP binding site that could be exploited for selective inhibitor design

• Species-specific editing domains that prevent mischarging of tRNA

• Unique insertion domains not present in the human enzyme

These structural differences are critical for developing selective inhibitors that target the bacterial enzyme without affecting the human counterpart, similar to the approach that might be taken with other N. farcinica virulence-associated proteins .

How can researchers design definitive experiments to characterize the kinetic mechanism of N. farcinica hisS?

Defining the precise kinetic mechanism of N. farcinica hisS requires a systematic experimental approach that distinguishes between ordered and random substrate binding, identifies rate-limiting steps, and quantifies individual rate constants. A comprehensive kinetic characterization includes:

• Initial velocity studies: Vary concentrations of all three substrates (histidine, ATP, and tRNAHis) individually while keeping others at saturating levels. Plot data using Lineweaver-Burk, Eadie-Hofstee, and Hanes-Woolf transformations to identify potential cooperativity or substrate inhibition.

• Product inhibition studies: Use AMP, PPi, and charged tRNAHis as product inhibitors to determine inhibition patterns, which can distinguish between ordered and random mechanisms.

• Pre-steady-state kinetics: Use rapid quench-flow or stopped-flow techniques to measure rates of adenylate formation and transfer to tRNA, identifying potential rate-limiting steps.

• Isotope exchange experiments: Measure PPi-ATP exchange to quantify the reversibility of the adenylate formation step.

The following table summarizes expected results for different mechanistic models:

These kinetic approaches can also help evaluate whether potential inhibitors target the first step (amino acid activation) or the second step (transfer to tRNA) of the aminoacylation reaction, critical information for rational drug design.

What critical residues determine substrate specificity and catalytic efficiency in N. farcinica hisS?

Identifying critical residues that determine substrate specificity and catalytic efficiency requires a systematic mutagenesis approach informed by structural studies and sequence alignment with related enzymes. The following methodological approach is recommended:

• Sequence alignment analysis: Compare N. farcinica hisS with well-characterized histidyl-tRNA synthetases from other organisms to identify conserved motifs and divergent regions. Pay particular attention to:

  • HIGH sequence motif (typically involved in histidine binding)

  • KMSKS loop (involved in ATP binding and transition state stabilization)

  • Discriminator base recognition elements

  • Editing domain residues

• Site-directed mutagenesis strategy: Generate single amino acid substitutions at predicted critical positions:

  • Conservative substitutions to assess the importance of specific chemical properties

  • Non-conservative substitutions to completely abolish function

  • Introduce residues from human histidyl-tRNA synthetase at non-conserved positions to assess species specificity

• Functional assessment of mutants: Measure both steps of the aminoacylation reaction separately:

  • Pyrophosphate exchange assay for amino acid activation

  • Complete aminoacylation assay for tRNA charging

Key residues likely to be critical include those involved in:

• Histidine side chain recognition (selectivity over other amino acids)

• ATP binding and catalysis of adenylate formation

• tRNAHis acceptor stem recognition (including the discriminator base)

• Positioning of the 3' end of tRNA for aminoacyl transfer

Understanding these critical residues provides foundational knowledge for the development of selective inhibitors that could target N. farcinica without affecting human histidyl-tRNA synthetase, potentially offering new therapeutic options for difficult-to-treat Nocardia infections .

How does recombinant N. farcinica hisS research contribute to understanding bacterial pathogenesis mechanisms?

Recombinant N. farcinica hisS research provides valuable insights into bacterial pathogenesis through multiple avenues:

• Protein synthesis regulation during infection: By studying hisS activity under conditions mimicking the host environment (oxidative stress, nutrient limitation, pH changes), researchers can understand how N. farcinica modulates protein synthesis during infection. This is particularly relevant given that N. farcinica can cause severe invasive infections with multiple abscesses affecting various organs, including the brain, lungs, and soft tissues .

• Stress response and adaptation: Comparing hisS activity under different stress conditions helps elucidate how N. farcinica adapts to host defense mechanisms. This is especially important since N. farcinica can trigger inflammatory responses, including activation of MAPK and NF-κB signaling pathways and production of TNF-α in macrophages .

• Virulence factor expression: Protein synthesis machinery, including hisS, is essential for the expression of virulence factors. Studying the correlation between hisS activity and virulence factor production (such as Nfa34810, which facilitates host cell invasion) can reveal regulatory mechanisms of pathogenesis .

• Persistence mechanisms: N. farcinica can establish persistent infections requiring prolonged antibiotic therapy. Understanding how hisS functions under antibiotic pressure may reveal mechanisms of persistence.

Methodological approaches should include:

• Examining hisS expression during different stages of infection using RT-qPCR

• Assessing hisS activity in cellular infection models

• Correlating hisS activity with the expression of known virulence factors like Nfa34810

• Investigating potential moonlighting functions of hisS beyond its canonical role in protein synthesis

What experimental approaches best evaluate N. farcinica hisS as a potential antimicrobial target?

Systematic evaluation of N. farcinica hisS as an antimicrobial target requires a multifaceted approach:

• Target validation studies:

  • Construct conditional knockdown strains to verify essentiality

  • Perform complementation studies with human histidyl-tRNA synthetase to confirm lack of functional redundancy

  • Assess growth kinetics and virulence of strains with reduced hisS expression

• High-throughput screening (HTS) assay development:

  • Develop a robust aminoacylation assay adaptable to 384-well format

  • Implement counterscreens against human histidyl-tRNA synthetase to identify selective inhibitors

  • Include orthogonal assays to confirm mechanism of action

• Structure-based drug design:

  • Perform fragment-based screening against crystallized N. farcinica hisS

  • Use molecular dynamics simulations to identify transiently open pockets

  • Design compounds that exploit structural differences between bacterial and human enzymes

• Lead compound evaluation:

  • Determine minimum inhibitory concentrations (MICs) against N. farcinica clinical isolates

  • Assess cytotoxicity against human cell lines

  • Evaluate efficacy in cellular infection models

The table below outlines expected characteristics of an ideal hisS inhibitor:

This approach is particularly promising given that N. farcinica infections can be difficult to treat, with some cases requiring prolonged antimicrobial therapy, especially in disseminated disease .

How can N. farcinica hisS research contribute to developing improved diagnostic methods for nocardiosis?

N. farcinica hisS research offers several avenues for improving nocardiosis diagnostics, addressing the current challenges in rapid and accurate identification of Nocardia species:

• Immunodiagnostic approaches:

  • Evaluate recombinant hisS as an antigen for antibody detection in patient sera

  • Develop anti-hisS monoclonal antibodies for antigen detection in clinical samples

  • Assess whether hisS elicits specific antibody responses during infection, similar to the immunodominant Nfa34810 protein

• Molecular diagnostic methods:

  • Design species-specific PCR primers targeting unique regions of the hisS gene

  • Develop multiplex PCR assays distinguishing N. farcinica from other Nocardia species

  • Create LAMP (Loop-mediated isothermal amplification) assays for point-of-care diagnostics

• Mass spectrometry-based approaches:

  • Identify hisS-derived peptide markers for inclusion in MALDI-TOF MS databases

  • Improve existing MALDI-TOF MS identification protocols, which have shown promise for rapid Nocardia species identification

  • Develop targeted proteomics assays (MRM/PRM) for direct detection in clinical samples

• Functional diagnostic assays:

  • Develop activity-based assays to detect aminoacylation activity in clinical samples

  • Create colorimetric or fluorescent indicators of hisS activity for rapid testing

The table below compares these different diagnostic approaches:

*After culture growth, which currently takes ≥3 days for Nocardia species

Improved diagnostics are crucial as early and accurate identification of N. farcinica could guide appropriate antimicrobial therapy, particularly important given its association with disseminated disease and potential antimicrobial resistance patterns .

What are the most common pitfalls in recombinant N. farcinica hisS expression and how can researchers overcome them?

Expression of recombinant N. farcinica hisS presents several technical challenges, many stemming from the organism's high G+C content and unique biology. Here are the most common pitfalls and their solutions:

• Low expression levels:

  • Problem: Codon bias due to N. farcinica's high G+C content (approximately 70%)

  • Solution: Use E. coli Rosetta or BL21-CodonPlus strains that supply rare tRNAs, or perform codon optimization of the gene sequence for the expression host

• Protein insolubility:

  • Problem: Formation of inclusion bodies

  • Solution: Lower induction temperature (16-18°C), reduce IPTG concentration (0.1-0.3 mM), co-express with chaperones (GroEL/GroES), or use solubility-enhancing fusion partners (SUMO, MBP)

• Protein instability:

  • Problem: Rapid degradation during expression or purification

  • Solution: Include protease inhibitors, add stabilizing agents (glycerol 10-20%, reducing agents), optimize buffer pH based on theoretical isoelectric point

• Low enzymatic activity:

  • Problem: Misfolding or loss of cofactors

  • Solution: Ensure buffer contains required metal ions (typically Mg²⁺, 2-5 mM), verify proper disulfide bond formation, refold protein if necessary

• Contamination with endotoxins:

  • Problem: Endotoxin interference with downstream applications, especially immunological studies

  • Solution: Include Triton X-114 extraction step or use endotoxin removal columns, consider expression in Gram-positive hosts

The table below presents a systematic troubleshooting approach for optimization:

Researchers should be aware that N. farcinica proteins may have unique folding requirements, as suggested by studies showing that proteins like Nfa34810 have specific localization (cell wall) and immunogenic properties that may impact recombinant expression .

How can researchers establish reliable enzymatic assays for N. farcinica hisS inhibitor screening?

Developing robust enzymatic assays for N. farcinica hisS inhibitor screening requires careful attention to assay design, validation, and quality control. Here's a comprehensive methodology:

• Primary assay development:

  • ATP-PPi exchange assay: Measures the first step of aminoacylation (amino acid activation)

  • tRNA aminoacylation assay: Measures complete reaction (charging of tRNA)

  • Pyrophosphate release assay: Monitors ATP hydrolysis using coupled enzyme systems

• Assay optimization parameters:

  • Buffer composition: Typically 50 mM HEPES pH 7.5, 10 mM MgCl₂, 50 mM KCl, 1 mM DTT

  • Enzyme concentration: Use 10-20% substrate conversion (typically 10-50 nM enzyme)

  • Substrate concentrations: Set at Km values for initial screening, then vary for mechanistic studies

  • Temperature and reaction time: 37°C, with time points at 5, 10, 15, and 30 minutes

• Assay validation metrics:

  • Z'-factor: Should exceed 0.7 for robust screening

  • Signal-to-background ratio: Minimum 5:1

  • Coefficient of variation: <15% across replicates

  • DMSO tolerance: Validate stability in 0.1-1% DMSO

• Control compounds:

  • Positive controls: Known aminoacyl-tRNA synthetase inhibitors (e.g., mupirocin)

  • Negative controls: Structurally related inactive compounds

  • Reference standards: Well-characterized inhibitors with defined IC₅₀ values

The following table outlines different assay formats suitable for inhibitor screening:

When implementing these assays, researchers should consider physiologically relevant conditions that mimic the host environment during N. farcinica infection, as studies have shown that the pathogen can activate specific signaling pathways and induce inflammatory responses that might influence enzyme activity in vivo .

What are critical considerations for data interpretation when studying N. farcinica hisS in the context of pathogenesis?

• Physiological relevance of experimental conditions:

  • Consider whether in vitro conditions reflect the in vivo environment

  • Account for changes in pH, temperature, oxidative stress, and nutrient availability during infection

  • Include controls that mimic conditions in different infection sites (lung, brain, skin) as N. farcinica can cause multiple types of infections

• Correlation between enzymatic activity and virulence:

  • Establish cause-effect relationships rather than mere correlations

  • Use genetically modified strains with controlled hisS expression levels

  • Compare results with known virulence factors like Nfa34810

• Host-pathogen interaction context:

  • Consider that N. farcinica activates specific immune pathways (MAPK, NF-κB) and triggers TNF-α production

  • Assess whether host factors modulate hisS activity

  • Evaluate hisS activity in the presence of host defense molecules

• Technical limitations awareness:

  • Recognize constraints of recombinant systems versus native protein studies

  • Account for differences between in vitro, ex vivo, and in vivo systems

  • Consider species-specific limitations of model systems

The table below presents a framework for data interpretation across different experimental contexts:

When interpreting results, researchers should particularly note that N. farcinica can cause disease in both immunocompromised and immunocompetent hosts, with varying clinical presentations from localized to disseminated infections . This host range diversity should inform experimental design and data interpretation, especially when considering hisS as a potential therapeutic target.

What novel methodologies might advance understanding of N. farcinica hisS structure-function relationships?

Emerging technologies offer promising avenues to deepen our understanding of N. farcinica hisS structure-function relationships, potentially revealing new insights into pathogenesis and drug development:

• Cryo-electron microscopy (Cryo-EM):

  • Enables visualization of hisS in complex with tRNA without crystallization

  • Allows capture of different conformational states during the catalytic cycle

  • Can reveal dynamic interactions that are difficult to observe in crystal structures

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Maps protein dynamics and conformational changes upon substrate binding

  • Identifies regions with differential solvent accessibility during catalysis

  • Provides insight into allosteric regulation mechanisms

• Single-molecule FRET:

  • Monitors real-time conformational changes during catalysis

  • Reveals heterogeneity in enzyme populations

  • Captures transient intermediates missed by ensemble techniques

• Deep mutational scanning:

  • Systematically assesses thousands of variants simultaneously

  • Maps complete fitness landscapes for enzyme function

  • Identifies non-obvious functional residues and epistatic interactions

• AlphaFold2 and related AI approaches:

  • Predicts protein structure with near-experimental accuracy

  • Enables in silico modeling of protein-protein and protein-ligand interactions

  • Facilitates virtual screening of potential inhibitors

These advanced methodologies could significantly accelerate our understanding of how N. farcinica hisS contributes to the pathogen's ability to invade host cells and cause severe infections, similar to the mechanistic insights gained about other virulence factors like Nfa34810 .

How might systems biology approaches integrate N. farcinica hisS research with broader pathogenesis studies?

Systems biology approaches offer powerful frameworks to integrate N. farcinica hisS research with broader pathogenesis studies, providing holistic insights into infection mechanisms:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data during infection

  • Correlate hisS expression/activity with global protein synthesis patterns

  • Map metabolism-virulence connections through flux balance analysis

• Network biology:

  • Construct protein-protein interaction networks including hisS

  • Identify hub proteins connecting translation machinery to virulence systems

  • Model information flow between pathogen systems during stress response

• Host-pathogen interactomics:

  • Map interactions between bacterial proteins (including hisS) and host factors

  • Identify potential moonlighting functions of hisS in host cells

  • Model competitive interactions for resources between host and pathogen

• In silico infection modeling:

  • Develop computational models of N. farcinica infection incorporating translation dynamics

  • Simulate effects of hisS inhibition on pathogen fitness in different host niches

  • Predict emergence of resistance mechanisms

The integration of these approaches would help contextualize hisS function within N. farcinica's broader virulence strategies, which include activation of specific host signaling pathways (like MAPK and NF-κB) and evasion of host immune responses, as demonstrated in studies of other N. farcinica virulence proteins .

What interdisciplinary collaborations would most accelerate translation of N. farcinica hisS research into clinical applications?

Accelerating the translation of N. farcinica hisS research into clinical applications requires strategic interdisciplinary collaborations spanning multiple fields:

• Structural biology and medicinal chemistry:

  • Structural biologists provide high-resolution structures of hisS

  • Medicinal chemists design and synthesize selective inhibitors

  • Computational chemists perform virtual screening and molecular dynamics simulations

• Microbiology and immunology:

  • Microbiologists assess inhibitor efficacy against diverse clinical isolates

  • Immunologists evaluate effects on host-pathogen interactions

  • Together they develop infection models that accurately reflect human disease

• Clinical medicine and diagnostic technology:

  • Infectious disease specialists identify unmet clinical needs

  • Diagnostic developers create rapid identification methods

  • Biomarker researchers evaluate hisS as a diagnostic or prognostic indicator

• Pharmaceutical sciences and regulatory affairs:

  • Formulation scientists optimize drug delivery

  • Pharmacologists determine pharmacokinetics/pharmacodynamics

  • Regulatory experts navigate approval pathways for orphan disease applications

A coordinated consortium approach would be particularly valuable given the relative rarity of Nocardia infections compared to other bacterial pathogens, allowing efficient resource utilization while addressing the substantial morbidity and mortality associated with disseminated nocardiosis .

Such interdisciplinary efforts could leverage insights from studies showing that N. farcinica can cause severe invasive infections in both immunocompromised and immunocompetent hosts , potentially leading to new therapeutic options for these challenging infections.