Recombinant Bartonella henselae 50S ribosomal protein L20 (rplT)

Basic Research Questions

• What is the function and significance of ribosomal protein L20 in Bartonella henselae?

  Ribosomal protein L20 (encoded by the rplT gene) is a critical component of the 50S ribosomal subunit in B. henselae. It serves two primary functions:

  • Structural role: L20 binds directly to 23S ribosomal RNA and is necessary for the in vitro assembly process of the 50S ribosomal subunit .

  • Regulatory role: L20 negatively regulates its own expression at the translational level through binding to specific RNA structures in the leader sequence of its mRNA .

  The protein is assembled at an early stage of ribosome assembly and is essential for proper ribosome biogenesis. Notably, L20 is not directly involved in the protein synthesizing functions of the 50S subunit once assembly is complete .

• How is recombinant B. henselae L20 protein expressed and purified for research applications?

  Expression and purification of recombinant B. henselae L20 involves several methodological steps:

  1. Gene cloning: The rplT gene can be amplified by PCR from B. henselae genomic DNA using primers designed based on the published sequence. The amplified fragment is then cloned into an expression vector (e.g., pET system for E. coli) .

  2. Expression system: Common expression systems include E. coli BL21(DE3) under control of a T7 or similar inducible promoter. Mammalian cell expression systems have also been used for producing recombinant Bartonella proteins with proper folding .

  3. Purification strategy:

     • Initial purification often employs affinity chromatography using tags such as His-tag or GST-tag

     • Further purification may include ion-exchange chromatography (taking advantage of L20's basic properties)

     • Final polishing is typically performed with size-exclusion chromatography

  4. Quality control: SDS-PAGE analysis typically shows a single band at approximately 15-18 kDa (depending on any tags used), with purity >85% achievable using optimized protocols .

  The purified protein should be stored with 5-50% glycerol at -20°C/-80°C for long-term stability, avoiding repeated freeze-thaw cycles .

• What expression vectors and host systems are most effective for producing recombinant B. henselae L20?

  Several expression systems have been successfully employed for recombinant Bartonella proteins:

  Expression System	Vector Examples	Advantages	Considerations
  E. coli BL21(DE3)	pET vectors, pACYC184	High yield, economical, well-established protocols	May form inclusion bodies, potential endotoxin contamination
  Mammalian cells	Proprietary vectors	Better folding for complex proteins, lower endotoxin	Higher cost, lower yield, longer production time
  Cell-free systems	Linear DNA templates	Avoids toxicity issues, rapid production	Expensive, smaller scale

  For basic research applications, E. coli systems are most commonly used. The pACYC184 vector has been successfully used for expressing Bartonella proteins as shown in several studies . When expressing L20 in E. coli, researchers should consider possible toxicity issues since overexpression of ribosomal proteins can disrupt the host cell's translation machinery.

  Special consideration should be given to codon optimization when expressing B. henselae genes in E. coli due to differences in codon usage between these bacterial species .

• How can researchers verify the functionality of recombinant B. henselae L20 protein?

  Verification of functional activity for recombinant L20 protein involves several complementary approaches:

  1. RNA binding assays:

     • Electrophoretic mobility shift assays (EMSA) with 23S rRNA fragments

     • Filter binding assays using radiolabeled RNA

     • Surface plasmon resonance (SPR) to measure binding kinetics

  2. Structural integrity verification:

     • Circular dichroism (CD) spectroscopy to confirm secondary structure

     • Limited proteolysis to assess proper folding

     • Thermal shift assays to determine stability

  3. Functional complementation:

     • Ability to rescue growth defects in bacterial strains with compromised L20 function

     • In vitro ribosome assembly assays using purified components

  4. Autoregulatory activity:

     • Reporter gene assays using the L20 leader sequence fused to a reporter gene

     • In vitro transcription-translation assays to demonstrate autoregulation

  A properly functional L20 protein should demonstrate specific binding to its target RNA sequences and the ability to participate in ribosome assembly processes .

Advanced Research Questions

• How does the L20-interacting RNA structure in B. henselae compare with other bacterial species?

  The L20-interacting RNA structure represents a conserved regulatory element found in many bacterial species, though with important variations:

  Feature	B. henselae	E. coli	B. subtilis
  Location	Leader sequence of infC-rpmI-rplT operon	Within rpmI coding sequence	Leader sequence of infC-rpmI-rplT-ysdA operon
  Mechanism	Likely transcription termination	Translation inhibition	Transcription termination
  RNA structure	Stem-loop with conserved L20 binding motif	Pseudoknot structure	Complex riboswitch-like arrangement
  Operon organization	Likely similar to other alphaproteobacteria	infC-rpmI-rplT	infC-rpmI-rplT-ysdA

  In B. subtilis, the system has been well characterized, showing that "L20 binds to a phylogenetically conserved domain and provokes premature transcription termination at the leader terminator" . The L20-binding RNA likely evolved through molecular mimicry of the L20 binding site on 23S rRNA.

  The structural details of B. henselae's L20-binding RNA have not been fully characterized, but comparative genomic analyses suggest conservation of this regulatory mechanism across proteobacteria .

• What role does L20 play in ribosome assembly defects observed in bacterial cold sensitivity?

  Ribosomal protein L20 plays a critical role in cold adaptation of bacterial ribosome assembly:

  1. Cold-sensitive phenotypes: Mutations affecting L20 or its regulatory elements can lead to cold-sensitive growth defects. This is because ribosome assembly is particularly vulnerable at lower temperatures where the kinetics of assembly are slower and more dependent on proper assembly factors .

  2. Evidence from suppressor studies: Research with BipA (a GTPase involved in ribosome assembly) showed that "exogenous expression of rplT restored the growth of bipA-deleted strain at low temperature by partially recovering the defects in ribosomal RNA processing and ribosome assembly" . This demonstrates L20's critical role in cold-temperature ribosome biogenesis.

  3. rRNA processing: L20 helps coordinate proper rRNA maturation, with defects in L20 function leading to "aberrant ribosome assembly" and "accumulation of precursor rRNA transcripts" . These defects are typically more pronounced at lower temperatures.

  4. Assembly pathway: L20 serves as an early assembly protein that creates binding sites for later assembly proteins in a hierarchical pathway. Disruption of this sequence at low temperatures can halt the entire assembly process .

  The relationship between L20 and cold sensitivity makes it a valuable target for studying ribosome assembly mechanisms and bacterial adaptation to environmental stresses.

• How can researchers use site-directed mutagenesis to study functional domains of B. henselae L20?

  Site-directed mutagenesis provides powerful insights into L20 structure-function relationships:

  1. Design strategy:

     • Target conserved residues identified through multiple sequence alignments

     • Focus on basic residues in the N-terminal domain likely involved in RNA binding

     • Create truncation mutants (ΔN and ΔC) to assess domain-specific functions

     • Design point mutations in regulatory regions that may affect autoregulation

  2. Methodology:

     • PCR-based mutagenesis using complementary primers containing the desired mutation

     • Subcloning into expression vectors like pACYC184 (successfully used for L20 studies)

     • Verification by DNA sequencing before expression

  3. Functional assessment:

     • RNA binding assays to assess affinity changes

     • In vivo complementation studies in L20-deficient strains

     • Reporter gene assays to measure effects on autoregulation

  4. Critical residues to target:

     • Based on studies in related proteins, the highly conserved basic residues in the N-terminal region

     • Residues at the protein-RNA interface identified from structural studies

     • Amino acids involved in potential protein-protein interactions

  This approach has been successfully applied to study ribosomal proteins, as seen in the study where "point mutations in rplT were introduced by site-directed mutagenesis PCR using pBIS02-2 as a template" .

• What approaches can be used to study L20's autoregulatory mechanism in B. henselae?

  Investigating L20's autoregulatory function requires specialized techniques:

  1. Reporter gene constructs:

     • Fusion of the L20 regulatory region to reporter genes (lacZ, GFP)

     • Example: "To generate a lacZ-fused regulatory region of the rpmI gene, the regulatory region of rpmI was amplified by PCR..."

     • Measurement of reporter expression under various L20 concentrations

  2. RNA structure probing:

     • In-line probing to identify RNA structural changes upon L20 binding

     • SHAPE (Selective 2'-hydroxyl acylation analyzed by primer extension)

     • Footprinting assays to map precise L20 binding sites

  3. In vitro transcription termination assays:

     • Reconstitution of the regulatory system with purified components

     • Analysis of L20-dependent transcription termination efficiency

  4. Mutational analysis:

     • Creating mutations in the leader RNA structure: "The M4 mutation is designed to destabilize the stem of the rho-independent terminator that forms upon L20 binding"

     • Testing the effects on regulation: "This mutation resulted in elevated constitutive expression"

  5. Physiological relevance:

     • Examining effects under stress conditions (temperature, antibiotics)

     • Correlation with ribosome assembly defects: "L20-interacting RNA mutants demonstrate improper rRNA processing at low temperatures"

  These approaches collectively provide a comprehensive understanding of how L20 regulates its own synthesis in response to cellular needs.

• How can recombinant B. henselae L20 be used for developing diagnostic tools for Bartonellosis?

  Recombinant L20 protein has potential applications in diagnostic assay development:

  1. Serological assays:

     • ELISA development using purified L20 to detect anti-Bartonella antibodies

     • Western blot confirmation tests for serodiagnosis

     • Potential sensitivity and specificity compared to other B. henselae antigens:

     Antigen	Sensitivity	Specificity	Notes
     rPap31	72%	61%	At cutoff value of 0.215
     rPap31-NTD	89%	56%	At cutoff value of 0.7985
     rPap31-NTD	39%	94%	At higher cutoff value of 1.366
     L20 (potential)	To be determined	To be determined	May offer complementary diagnostic value

  2. PCR-based detection:

     • Design of primers targeting conserved regions of the rplT gene

     • Development of multiplex PCR assays including rplT alongside other genetic markers

     • Potential for improving strain typing beyond current methods: "Our data are consistent with published evidence and with previous suggestions of intragenomic rearrangements in the type strain and suggest that human isolates come from a limited subset of B. henselae strains"

  3. Advantages of rplT as a diagnostic target:

     • Conservation across Bartonella species allows genus-level detection

     • Species-specific variations could enable discrimination between Bartonella species

     • As an essential gene, less likely to be lost during infection

  Development of these diagnostic applications requires thorough validation against gold standard methods and testing with diverse clinical samples.

• How does the genetic diversity of rplT compare across B. henselae isolates from different sources?

  The genetic diversity of B. henselae strains and its impact on rplT sequences:

  1. Observed patterns of diversity:

     • "A study of 59 isolates of Bartonella henselae reveals relatively limited diversity among those of human origin (n = 28)"

     • Human isolates show more genetic homogeneity compared to feline isolates

     • Studies indicate "limited diversity among human isolates (including imported strains) from the majority of feline isolates"

  2. Methodological approaches to assess diversity:

     • Multi-locus sequence typing (MLST): "MLST was applied to a larger collection of feline and human B. henselae isolates from Europe, Australia and the USA"

     • PCR-RFLP analysis of specific genetic loci

     • Whole genome sequencing for comprehensive analysis

  3. Implications for research:

     • Selection of representative strains for functional studies of L20

     • Understanding potential functional variations in L20 across strain types

     • Development of universal primers for amplification of rplT from diverse isolates

  4. Clinical relevance:

     • Predominant sequence types in human infections: "ST1 might possess additional virulence factors, which could encode for a more effective transmission from cats to humans, or a better survival of the pathogen in the human host"

     • Geographic distribution patterns: "Human isolates from all over Eastern Australia were most commonly 16S rDNA (Bergmans) type I"

  This diversity data is essential for designing broadly applicable recombinant L20 proteins for research and diagnostic applications.

• What are the experimental approaches to study interactions between B. henselae L20 and 23S rRNA?

  Several complementary approaches can characterize L20-23S rRNA interactions:

  1. RNA-protein binding assays:

     • Electrophoretic mobility shift assays (EMSA) with purified components

     • Filter binding assays with labeled RNA fragments

     • Surface plasmon resonance (SPR) for binding kinetics determination

     Method	Advantages	Limitations	Typical Buffer Conditions
     EMSA	Visualizes complex formation, simple setup	Semi-quantitative, potential dissociation during electrophoresis	10 mM Tris-HCl pH 7.5, 50-100 mM KCl, 5-10 mM MgCl₂
     Filter binding	Quantitative, determines Kd values	Requires radiolabeled RNA	20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl₂, 5% glycerol
     SPR	Real-time kinetics, label-free	Expensive equipment, surface immobilization may affect binding	10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant

  2. Structural analysis:

     • RNA footprinting to identify protected nucleotides

     • Structural studies (X-ray crystallography, cryo-EM) of the L20-rRNA complex

     • Computational modeling based on known structures

  3. Mutational analysis:

     • Site-directed mutagenesis of key residues in L20

     • Mutations in 23S rRNA binding sites

     • Analysis of effects on binding affinity and ribosome assembly

  4. In vivo approaches:

     • RNA-protein crosslinking followed by immunoprecipitation (CLIP)

     • Genetic complementation studies with mutant L20 variants

     • Analysis of effects on ribosome assembly and function

  These approaches would build on existing knowledge that "L20 binds directly to 23S ribosomal RNA and is necessary for the in vitro assembly process of the 50S ribosomal subunit" .

• How can recombinant L20 be used to study ribosome assembly mechanisms in B. henselae?

  Recombinant L20 provides valuable tools for studying ribosome assembly:

  1. In vitro reconstitution assays:

     • Step-wise assembly of ribosomal components using purified recombinant proteins

     • Fluorescence-based monitoring of assembly kinetics

     • Analysis of assembly intermediate structures by cryo-EM

  2. Suppressor studies:

     • Use of recombinant L20 to rescue assembly defects in other ribosomal protein mutants

     • Example: "Exogenous expression of rplT restored the growth of bipA-deleted strain at low temperature by partially recovering the defects in ribosomal RNA processing and ribosome assembly"

     • Quantification of rescue efficiency under different conditions

  3. Temperature-dependent assembly analysis:

     • Comparison of assembly kinetics at different temperatures (20°C vs. 37°C)

     • Analysis of L20's role in cold-adaptation of ribosome assembly

     • Correlation with rRNA processing defects: "L20-interacting RNA mutants demonstrate improper rRNA processing at low temperatures"

  4. Protein-protein interaction studies:

     • Identification of L20's interaction partners during assembly

     • Pull-down assays using tagged recombinant L20

     • Characterization of assembly intermediates containing L20

  5. FRET-based assembly monitoring:

     • Fluorescently labeled L20 and other ribosomal components

     • Real-time monitoring of assembly events

     • Quantification of assembly rates and efficiency

  These approaches provide mechanistic insights into B. henselae ribosome assembly, particularly the early stages where L20 plays a critical role.

• What is the potential role of B. henselae L20 in pathogenesis and host interactions?

  While primarily known for ribosomal functions, L20 may contribute to pathogenesis:

  1. Potential extracellular roles:

     • Ribosomal proteins can have moonlighting functions outside the ribosome

     • Possible interaction with host molecules during infection

     • Potential immunomodulatory effects during B. henselae infection

  2. Contribution to stress adaptation:

     • L20's role in ribosome assembly under stress conditions may impact virulence

     • Adaptation to temperature changes during zoonotic transmission (feline 39°C → human 37°C)

     • Response to other host-associated stresses (pH, nutrient limitation)

  3. Host immune recognition:

     • Potential recognition of L20 by the adaptive immune system

     • Development of anti-L20 antibodies during infection: "several researchers have successfully isolated B. henselae strains that lack a PCR-detectable heme-binding protein" , suggesting antigenic variation in certain proteins

     • Possible diagnostic value of anti-L20 antibodies

  4. Translation regulation of virulence factors:

     • Impact of L20 levels on expression of pathogenicity determinants

     • Effect on bacterial growth kinetics and persistence in host tissues

  5. Experimental approaches:

     • Detection of L20 in infected host cells

     • Analysis of antibody responses to L20 in infected hosts

     • Effects of L20 mutations on bacterial survival in cellular infection models

  Understanding these potential roles would provide new insights into B. henselae pathogenicity and host interaction mechanisms.

• How can computational approaches aid in studying the structure and function of B. henselae L20?

  Computational methods provide valuable tools for L20 research:

  2. RNA-protein interaction prediction:

     • Docking simulations of L20 with 23S rRNA fragments

     • Electrostatic potential mapping to identify RNA-binding surfaces

     • Prediction of binding affinity changes with mutations

  3. Comparative genomics:

     • Multiple sequence alignments to identify conserved residues across Bartonella species

     • Analysis of selection pressure on different L20 domains

     • Identification of species-specific variations that might relate to host adaptation

  4. Systems biology approaches:

     • Integration of L20 into ribosome assembly network models

     • Simulation of assembly kinetics under different conditions

     • Prediction of phenotypic effects of L20 mutations

  5. Data visualization tools:

     • Structure visualization with PyMOL or ChimeraX

     • Network visualization of L20 interactions

     • Integration of experimental data with structural models

  These computational approaches complement experimental studies and can guide hypothesis generation for further investigation of B. henselae L20 function.