Recombinant Bartonella henselae Cysteine--tRNA ligase (cysS), partial

What is the biological role of Cysteine--tRNA ligase in Bartonella henselae?

Cysteine--tRNA ligase (cysS) in Bartonella henselae catalyzes the attachment of cysteine to its cognate tRNA molecule (tRNACys), an essential step in protein synthesis. This aminoacylation reaction is critical for translational fidelity and ensures accurate incorporation of cysteine into growing polypeptide chains. In B. henselae, an intracellular pathogen causing cat scratch disease and other clinical manifestations, proper protein synthesis is particularly important for bacterial survival within host cells and virulence factor expression . The enzyme belongs to the broader family of aminoacyl-tRNA synthetases that maintain translational accuracy across all domains of life.

How does Bartonella henselae cysS compare to homologous enzymes in other bacterial species?

B. henselae cysS shares structural and functional similarities with homologs in other alpha-proteobacteria, though with distinct features reflecting its evolution as an intracellular pathogen. Unlike free-living bacteria with more complex tRNA modification systems, B. henselae has undergone substantial reduction in its tRNA modification genes as part of its adaptation to intracellular lifestyle . Comparative genomic analyses have revealed that B. henselae encodes 26 genes responsible for 23 distinct tRNA modifications, compared to more extensive modification systems in non-pathogenic bacteria . These specific adaptations in tRNA processing machinery, including cysS, likely contribute to B. henselae's specialized translational needs during infection cycles.

What is known about the regulation of cysS expression in Bartonella henselae?

Expression of cysS in B. henselae appears to be regulated by multiple factors, including the stringent response (SR) and the BatR/BatS two-component system . Research indicates that the SR components DksA and SpoT, along with the alternative sigma factor RpoH1, play critical roles in the coordinated expression of various B. henselae virulence factors during host infection. The SR signaling and the physiological pH-induced BatR/BatS TCS likely control the spatiotemporal expression of adaptation factors, potentially including cysS . This complex regulatory network ensures appropriate protein synthesis machinery activity during different stages of the bacterial life cycle, particularly during transitions between arthropod vectors and mammalian hosts.

What expression systems have proven most effective for recombinant production of Bartonella henselae cysS?

E. coli-based expression systems remain the predominant platform for recombinant B. henselae cysS production, with BL21(DE3) strains being particularly effective. Vector selection typically favors pET-series vectors with N-terminal His-tags for simplified purification . Critical optimization parameters include:

Codon optimization for E. coli expression is recommended as B. henselae exhibits different codon usage patterns, particularly for rare codons encoding arginine and leucine residues.

What purification strategies yield the highest purity and activity of recombinant B. henselae cysS?

A multi-step purification approach is recommended for obtaining high-purity, active recombinant B. henselae cysS. The optimal protocol involves:

• Initial capture using Ni-NTA affinity chromatography (for His-tagged constructs) with imidazole gradient elution (50-300 mM)

• Intermediate purification via ion-exchange chromatography (typically Q-Sepharose at pH 8.0)

• Final polishing step using size-exclusion chromatography in buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl₂, and 5 mM DTT

Buffer optimization studies indicate that maintaining reducing conditions throughout purification (2-5 mM DTT or 1 mM TCEP) is critical for preserving enzymatic activity, as the enzyme contains catalytically important cysteine residues that are susceptible to oxidation . Purification under these conditions typically yields protein with >90% purity suitable for biochemical and structural studies.

What are the major challenges in expressing functional B. henselae cysS and how can they be overcome?

The primary challenges in recombinant expression of functional B. henselae cysS include:

• Protein solubility issues: The enzyme tends to form inclusion bodies at high expression levels. This can be mitigated by:

  • Expression at lower temperatures (18-20°C)

  • Co-expression with molecular chaperones (GroEL/GroES or DnaK/DnaJ/GrpE systems)

  • Fusion with solubility-enhancing tags such as MBP or SUMO

• Catalytic activity preservation: B. henselae cysS contains critical cysteine residues sensitive to oxidation. Preservation strategies include:

  • Maintaining reducing conditions throughout purification

  • Adding zinc or other divalent metal ions (0.1-1 mM) to stabilize the protein structure

  • Purification under anaerobic conditions for highest activity retention

• Protein stability issues: The enzyme shows reduced stability in solution after purification. This can be addressed by:

  • Addition of glycerol (10-20%) to storage buffers

  • Flash-freezing in liquid nitrogen rather than slow freezing

  • Storage at higher concentrations (>1 mg/ml) to reduce surface denaturation effects

Implementation of these strategies has been shown to increase functional protein yields by 3-4 fold compared to standard protocols .

What methodologies provide the most sensitive and accurate assessment of B. henselae cysS aminoacylation activity?

Several complementary assays can be employed for comprehensive characterization of B. henselae cysS activity:

• Aminoacylation assay using radioactive substrates: The gold standard approach involves monitoring the incorporation of [³⁵S]-cysteine or [¹⁴C]-cysteine into tRNACys. This assay provides high sensitivity with detection limits in the picomolar range. The reaction typically contains:

  • Purified recombinant cysS (10-100 nM)

  • Total tRNA or purified tRNACys (1-10 μM)

  • Labeled cysteine (5-20 μM)

  • ATP (2-5 mM)

  • Mg²⁺ (5-10 mM)

  • Buffer at physiological pH (7.2-7.5)

• Pyrophosphate release assay: This non-radioactive alternative measures the pyrophosphate released during the aminoacylation reaction using coupled enzyme systems and fluorescence or absorbance detection.

• tRNA mobility shift assay: Based on the differential electrophoretic mobility of charged versus uncharged tRNAs under acidic conditions, providing qualitative confirmation of aminoacylation.

For kinetic parameter determination, the enzyme should be characterized under steady-state conditions with varying substrate concentrations. This typically reveals Km values for cysteine in the range of 5-50 μM and for tRNACys in the range of 0.5-2 μM, with kcat values typically between 1-10 s⁻¹ .

How can researchers assess the tRNA substrate specificity of B. henselae cysS?

Determining tRNA substrate specificity requires:

• Comparative aminoacylation assays with:

  • Homologous B. henselae tRNACys

  • Heterologous tRNACys from other organisms

  • Other tRNA species as negative controls

• Identity element mapping through:

  • Site-directed mutagenesis of conserved elements in tRNACys

  • In vitro transcription of variant tRNAs

  • Aminoacylation assays with variant substrates

• Cross-aminoacylation studies to assess the enzyme's ability to charge tRNACys from different species

Research has shown that B. henselae cysS, like other bacterial cysteinyl-tRNA synthetases, primarily recognizes the U73 discriminator base and the G15:G48 tertiary base pair as identity elements . Interestingly, the reduced tRNA modification profile of B. henselae compared to free-living bacteria may influence the recognition specificity, as certain tRNA modifications can serve as identity or anti-identity elements for aminoacyl-tRNA synthetases.

What approaches enable accurate determination of inhibitor effectiveness against B. henselae cysS?

Inhibitor characterization requires a systematic approach:

• IC₅₀ determination using:

  • Standard aminoacylation assays with varying inhibitor concentrations

  • Controls to distinguish between specific inhibition and non-specific effects

• Mechanism of inhibition studies through:

  • Kinetic analysis with varying substrate and inhibitor concentrations

  • Lineweaver-Burk or other double-reciprocal plots for mechanism determination

  • Global fitting of data to different inhibition models

• Direct binding analysis using:

  • Isothermal titration calorimetry (ITC)

  • Surface plasmon resonance (SPR)

  • Microscale thermophoresis (MST)

• Thermal shift assays to assess inhibitor-induced stabilization effects

When testing potential inhibitors, researchers should consider compounds targeting different stages of the aminoacylation reaction: (1) ATP binding, (2) cysteine activation, (3) cysteine transfer to tRNA, or (4) product release. Natural product screening has identified several promising inhibitor classes, including certain sulfamoyl analogues and aminoacyl-adenylate mimics with IC₅₀ values in the low micromolar range .

What structural domains characterize B. henselae cysS and how do they contribute to enzymatic function?

Although the complete crystal structure of B. henselae cysS has not been reported, comparative structural analysis with homologous bacterial cysteinyl-tRNA synthetases suggests a multi-domain architecture with distinct functional regions:

• Catalytic domain (N-terminal): Contains the HIGH and KMSKS motifs characteristic of class I aminoacyl-tRNA synthetases, forming the active site for ATP binding and cysteine activation

• Anticodon-binding domain (C-terminal): Responsible for specific recognition of the tRNACys anticodon loop

• Editing domain: Contains the zinc-binding motif for hydrolyzing mis-acylated Cys-tRNACys, thereby enhancing translational fidelity

Each domain contains conserved sequence motifs that contribute to specific aspects of the aminoacylation reaction. The catalytic domain contains the signature sequence motifs of class I aminoacyl-tRNA synthetases (HIGH and KMSKS), while the anticodon-binding domain contains residues specific for tRNACys recognition . These structural features reflect B. henselae's evolutionary adaptation as an intracellular pathogen, potentially with specialized mechanisms for maintaining translational accuracy under the stress conditions encountered during infection.

How can researchers address challenges in crystallizing B. henselae cysS for structural studies?

Crystallization of B. henselae cysS presents several challenges that can be addressed through the following approaches:

• Construct optimization:

  • Generate truncation variants to remove flexible regions

  • Create fusion constructs with crystallization chaperones (e.g., T4 lysozyme)

  • Design surface entropy reduction mutants to promote crystal contacts

• Sample preparation optimization:

  • Ensure high protein purity (>95%) through additional purification steps

  • Verify protein homogeneity by dynamic light scattering

  • Use thermal shift assays to identify stabilizing buffer conditions

• Crystallization condition screening:

  • Employ commercial sparse matrix screens for initial hits

  • Use additive screens to optimize initial crystallization conditions

  • Explore co-crystallization with substrates, substrate analogs, or inhibitors

• Alternative approaches when crystallization proves challenging:

  • Cryo-electron microscopy for structural determination without crystals

  • Small-angle X-ray scattering for low-resolution shape information

  • Hydrogen-deuterium exchange mass spectrometry for dynamics information

  • Molecular modeling based on homologous structures

Researchers have found that co-crystallization with tRNACys or non-hydrolyzable ATP analogs often stabilizes the enzyme in a defined conformation, facilitating crystal formation. Additionally, inclusion of zinc or other divalent metal ions in crystallization buffers can enhance structural stability and crystal quality .

How do post-translational modifications affect B. henselae cysS function and structure?

Post-translational modifications (PTMs) of B. henselae cysS may play significant roles in regulating its activity and stability, though these remain understudied compared to modifications of its tRNA substrates. Key aspects include:

• Oxidative modifications:

  • Catalytically important cysteine residues in the active site are susceptible to oxidation

  • Reversible oxidation may serve as a regulatory mechanism during oxidative stress

  • Irreversible oxidation can lead to enzyme inactivation

• Phosphorylation:

  • Potential phosphorylation sites have been predicted in the connecting peptide regions

  • These modifications may influence domain orientation and substrate binding

  • The BatR/BatS two-component system may regulate cysS activity through phosphorylation cascades

• Potential regulatory implications:

  • PTMs may fine-tune enzyme activity in response to changing conditions during infection

  • Host-induced stress might trigger specific modifications affecting aminoacylation efficiency

  • PTM patterns could differ between the vector and mammalian host environments

The study of these modifications requires mass spectrometry-based proteomic approaches, including enrichment techniques for specific PTMs and careful sample preparation to preserve labile modifications. Comparing PTM profiles under different growth conditions may reveal regulatory mechanisms specific to the B. henselae infection cycle .

How does B. henselae cysS contribute to bacterial survival during infection?

B. henselae cysS plays several critical roles during infection:

• Maintaining protein synthesis under stress conditions:

  • During host infection, B. henselae encounters various stress conditions including oxidative stress, nutrient limitation, and pH changes

  • Efficient aminoacylation maintains translational capacity under these adverse conditions

  • The enzyme may have evolved specialized features for function within the intracellular environment

• Supporting virulence factor expression:

  • Proper aminoacylation is essential for accurate translation of key virulence factors

  • The VirB/D4 type IV secretion system, critical for B. henselae pathogenesis, requires precise protein synthesis for assembly and function

  • BadA protein, an important adhesin for bacterial attachment to host cells, relies on accurate translation

• Adaptation to host environments:

  • The stringent response, which regulates B. henselae virulence, may modulate cysS activity to adjust protein synthesis rates

  • Differential regulation during transitions between arthropod vectors and mammalian hosts ensures appropriate protein synthesis capacity

These functions highlight the potential of cysS as both a virulence contributor and a potential antimicrobial target for treating B. henselae infections like cat scratch disease .

What evidence suggests B. henselae cysS could serve as a potential therapeutic target?

Several lines of evidence support B. henselae cysS as a promising therapeutic target:

• Essential enzymatic function:

  • As an aminoacyl-tRNA synthetase, cysS performs a non-redundant, essential function in protein synthesis

  • Inhibition would block bacterial protein synthesis without immediate substitution pathways

  • The enzyme's essential nature is supported by genomic analyses showing retention of cysS even in the reduced genome of B. quintana

• Structural divergence from human homologs:

  • Bacterial cysteinyl-tRNA synthetases differ significantly from their eukaryotic counterparts

  • The active site architecture and tRNA recognition mechanisms present exploitable differences

  • These structural differences enable selective targeting of the bacterial enzyme

• Involvement in stress response and virulence:

  • The enzyme may be upregulated during infection and stress conditions

  • Connection to stringent response pathways suggests a role in adaptation to host environments

  • Inhibiting cysS could attenuate bacterial survival under the stress conditions of infection

• Potential for multi-target inhibition:

  • Compounds targeting conserved features of aminoacyl-tRNA synthetases might address multiple Bartonella species

  • This is particularly relevant given the diverse clinical manifestations of Bartonellosis, from cat scratch disease to endocarditis

Development of selective inhibitors would require detailed structural information and high-throughput screening approaches, potentially leveraging the recombinant protein for drug discovery campaigns .

How does B. henselae tRNA modification machinery interact with cysS function during infection?

The interaction between B. henselae tRNA modification systems and cysS function presents a fascinating area of research with implications for bacterial pathogenesis:

• Coordinated regulation and expression:

  • Both tRNA modification enzymes and cysS appear regulated by similar stress response pathways

  • Stringent response components (DksA, SpoT) and alternative sigma factors (RpoH1) likely coordinate expression of these translation-related functions

  • This co-regulation ensures balanced production of charged, properly modified tRNAs

• Functional interdependence:

  • Research has shown that B. henselae has undergone substantial reduction in tRNA modification genes compared to free-living bacteria, retaining 26 genes for 23 distinct modifications

  • These specific modifications may influence cysS recognition of tRNACys and charging efficiency

  • The modified nucleosides at positions 34 and 37 can affect tRNA structure and aminoacylation rates

• Adaptation to intracellular lifestyle:

  • The streamlined tRNA modification system of B. henselae reflects its adaptation to the intracellular niche

  • This specialized modification pattern may allow cysS to function optimally within the specific biochemical environment of host cells

  • The balance between modification simplification and translational fidelity represents an evolutionary compromise

• Pathogenesis implications:

  • Disruption of this delicate balance between tRNA modification and aminoacylation could affect bacterial survival and virulence

  • Targeting both systems simultaneously might provide synergistic antimicrobial effects

  • Understanding these interactions could reveal new aspects of B. henselae pathogenesis

This area remains a promising frontier for research, potentially revealing novel aspects of how translation-related processes adapt during host-pathogen interactions .

How can recombinant B. henselae cysS be utilized for development of novel diagnostic approaches?

Recombinant B. henselae cysS offers several promising applications for improving diagnosis of Bartonellosis:

• Serological test development:

  • As a conserved bacterial protein, recombinant cysS can serve as an antigen for detecting anti-Bartonella antibodies

  • ELISA and Western blot assays using purified recombinant cysS could complement existing diagnostic approaches

  • This may address limitations of current serological tests that show variable sensitivity (72-89%) and specificity (56-94%)

• Aptamer-based detection systems:

  • Selection of DNA or RNA aptamers against recombinant cysS could enable development of rapid diagnostic tests

  • Aptamer-based electrochemical biosensors might provide point-of-care testing options

  • Such approaches could overcome the challenges of current diagnostic methods that often require specialized laboratory facilities

• Molecular beacon probes:

  • Designing probes that interact specifically with cysS could enable detection of B. henselae in clinical samples

  • This approach might improve upon current PCR methods targeting the 16S rRNA gene or other targets

  • Combining multiple molecular targets including cysS could enhance diagnostic specificity

• Metabolic activity assays:

  • Leveraging cysS enzymatic function for detecting viable bacteria in clinical samples

  • This functional approach could distinguish between active infection and past exposure

The development of such diagnostic tools could address current challenges in Bartonellosis diagnosis, particularly for atypical presentations or in patients with negative serology despite clinical suspicion .

What approaches can uncover the role of B. henselae cysS in bacterial persistence during chronic infection?

Understanding cysS contribution to bacterial persistence requires multi-faceted approaches:

• Conditional expression systems:

  • Development of inducible/repressible cysS expression systems in B. henselae

  • Titrating expression levels to determine minimal requirements for persistence

  • Temporal control of expression to assess stage-specific requirements during infection

• Time-resolved proteomics:

  • Analysis of cysS expression and modifications throughout infection cycle

  • Correlation with global proteome changes during acute versus persistent infection

  • Identification of co-regulated proteins that might function together with cysS

• Single-cell analyses:

  • Investigating potential heterogeneity in cysS expression within bacterial populations

  • Determining if subpopulations with distinct cysS expression patterns exhibit different persistence properties

  • Correlating expression with bacterial metabolic state and replication rates

• Animal model studies:

  • Using engineered B. henselae strains with tagged or modified cysS

  • Tracking bacterial distribution and persistence in various host tissues

  • Testing persistence capabilities of strains with altered cysS activity or regulation

These approaches could provide insights into how aminoacylation capacity relates to the ability of B. henselae to establish persistent infections, which underlie conditions ranging from asymptomatic bacteremia to chronic manifestations like endocarditis .

How can comparative analysis of cysS across Bartonella species inform evolutionary adaptation to different hosts?

Comparative analysis of cysS across Bartonella species provides a window into pathogen evolution:

• Sequence and structural comparison:

  • Alignment of cysS sequences from various Bartonella species (B. henselae, B. quintana, B. clarridgeiae, etc.)

  • Identification of conserved versus variable regions that might reflect host-specific adaptations

  • Particular focus on differences between species infecting distinct mammalian hosts (cats, humans, rodents)

• Functional characterization:

  • Comparative biochemical analysis of recombinant cysS from different Bartonella species

  • Assessment of kinetic parameters, substrate specificity, and inhibitor sensitivity

  • Correlation of functional differences with host range and pathogenicity patterns

• Genomic context analysis:

  • Examination of cysS gene neighborhoods across Bartonella species

  • Identification of potential operon structures or co-regulated genes

  • Correlation with observed gene decay patterns, as seen in B. quintana compared to B. henselae

• Selection pressure analysis:

  • Calculation of dN/dS ratios to identify sites under positive or purifying selection

  • Mapping of these sites to functional domains of the enzyme

  • Correlation with host-specific adaptation patterns