Recombinant Bartonella quintana Translation initiation factor IF-1 (infA)

What is Bartonella quintana Translation initiation factor IF-1 and what is its significance in bacterial protein synthesis?

Translation initiation factor IF-1 (encoded by the infA gene) is a critical component of the bacterial translational apparatus that functions during the initiation phase of protein synthesis. While most research on bacterial IF-1 has been conducted in model organisms like Escherichia coli, the fundamental role is likely conserved in B. quintana. Studies in E. coli have demonstrated that IF-1 is essential for cell viability, as disruption of the chromosomal infA gene leads to cell death unless the gene is expressed in trans from a plasmid . The significance of IF-1 is underscored by observations that cells depleted of this factor exhibit few polysomes, confirming its crucial function in translation initiation .

In B. quintana specifically, the infA gene likely plays a similar essential role, though this pathogen faces unique challenges as it must adapt to diverse host environments. As an intracellular bacterium that infects human erythrocytes and vascular endothelial cells, B. quintana requires precisely regulated protein synthesis to survive within these different cellular niches .

What expression systems are most effective for producing recombinant B. quintana IF-1?

For recombinant expression of B. quintana IF-1, several prokaryotic and eukaryotic expression systems may be considered, with methodological considerations for each:

E. coli-based expression systems:

• BL21(DE3) strain with pET vector systems allows for high-yield expression under IPTG induction

• Codon optimization is recommended as B. quintana may have different codon usage patterns than E. coli

• Expression conditions: typically 18-25°C post-induction to minimize inclusion body formation

• Co-expression with chaperones (e.g., GroEL/GroES) may enhance solubility

Cell-free protein synthesis:

• Enables rapid screening of expression conditions

• Avoids potential toxicity issues that might occur in living cells

• Can incorporate non-canonical amino acids for functional studies

Given the small size of bacterial IF-1 proteins (typically ~70 amino acids), purification tags such as 6xHis should be compatible without significantly altering protein function. Verification of proper folding should be conducted using circular dichroism or functional assays.

How might infA expression patterns contribute to B. quintana's adaptive strategies during infection?

B. quintana demonstrates remarkable adaptability to diverse host environments, persisting in both human and non-human primate hosts. The regulation of essential genes like infA may play a role in this adaptability, though this remains to be specifically investigated.

Research methodology to explore this question:

• Transcriptomic profiling (RNA-seq) of B. quintana under various growth conditions mimicking different host environments

• Quantitative PCR to measure infA expression during different phases of infection

• Translational regulation studies using reporter constructs fused to the infA promoter and regulatory regions

• Conditional depletion systems to assess bacterial fitness in different host cell types when infA levels are altered

This approach is supported by existing research on B. quintana adaptation mechanisms. For instance, studies have shown that B. quintana can modify its surface proteins during prolonged bloodstream infection. The variably expressed outer-membrane proteins (Vomp) family undergoes changes including gene deletion during chronic infection, potentially to evade host immune responses . Similar adaptive regulation might occur with fundamental cellular machinery genes like infA in response to host pressures.

What is the relationship between infA function and the unique virulence mechanisms of B. quintana?

The relationship between translation initiation and virulence in B. quintana represents an intriguing research direction. While direct evidence linking infA to specific virulence mechanisms is not established in the provided literature, several methodological approaches could elucidate these connections:

• Comparative genomic analysis: Examine infA sequence conservation across strains with different virulence profiles

• Mutagenesis studies: Create conditional infA mutants to assess effects on:

  • Adhesion to host cells

  • Intracellular survival rates

  • Expression of known virulence factors like Vomp family proteins

B. quintana possesses several virulence mechanisms that could potentially be influenced by translation initiation efficiency:

• Anti-apoptotic functions mediated by genes like bepA

• Adhesion to erythrocytes facilitated by genes such as trwL2, trwL4, and trwL6

• Autoaggregation and collagen binding mediated by the Vomp family proteins

The Vomp family provides a particularly relevant model for studying protein expression regulation in B. quintana. Research has demonstrated that these proteins undergo variable expression during chronic infection, with some genes being deleted entirely. For example, VompA mediates autoaggregation while VompC confers collagen IV binding capability . Understanding how translation initiation factors influence the expression of such virulence determinants could provide insights into infection dynamics.

What structural and functional differences exist between IF-1 from B. quintana isolated from different host species?

B. quintana has been isolated from both humans and non-human primates, including Japanese macaques (Macaca fuscata) and rhesus macaques. Genomic analyses have revealed strain-specific differences that likely represent host-associated evolution .

Research methodology to investigate IF-1 differences:

• Comparative sequence analysis of infA across strains from different hosts

• Expression and purification of recombinant IF-1 from different strains

• Functional assays measuring translation initiation efficiency

• Binding studies with ribosomal components and other translation factors

• Crystallography to identify structural variations

Existing research has shown that Japanese macaque strains of B. quintana exhibit significant genomic differences compared to human strains, including a major chromosomal inversion and the absence of specific virulence genes . These large-scale genomic rearrangements might affect the regulation of fundamental cellular processes, potentially including translation initiation.

How can recombinant B. quintana IF-1 be utilized in developing diagnostic tools for blood culture-negative endocarditis?

B. quintana is recognized as a causative agent of blood culture-negative endocarditis (BCNE), a condition that presents significant diagnostic challenges . Current detection methods include immunofluorescent antibody (IFA) tests for IgG and IgM antibodies and PCR-based detection of B. quintana-specific genes from heart valve tissue .

Potential applications of recombinant IF-1 in diagnostic development:

• Serological assays: Recombinant IF-1 could serve as an antigen in ELISA or lateral flow assays to detect B. quintana-specific antibodies

  • Methodology: Express and purify tagged recombinant IF-1, validate with known positive sera, determine sensitivity and specificity

• Nucleic acid amplification: Primers targeting the infA gene region could enhance molecular detection

  • Methodology: Design primers specific to B. quintana infA, validate against related species, establish limits of detection

• Aptamer-based detection: Selection of aptamers against recombinant IF-1 for rapid diagnostics

  • Methodology: SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to identify high-affinity aptamers, develop colorimetric or fluorescent detection platforms

The diagnostic challenges of B. quintana infection are well-documented. In one study of 50 BCNE patients, only one patient tested positive for B. quintana by PCR of valve tissue samples, highlighting the need for improved diagnostic approaches .

What are the recommended protocols for efficiently cloning and expressing the B. quintana infA gene?

The following methodological approach is recommended for cloning and expressing B. quintana infA:

• Gene synthesis or PCR amplification:

  • Design primers with appropriate restriction sites for subsequent cloning

  • Include a Kozak sequence (GCCACC) immediately before the start codon

  • Consider codon optimization for the expression host

• Vector selection:

  • pET vectors (e.g., pET-28a) for bacterial expression with N- or C-terminal His-tags

  • Cold-shock expression vectors (e.g., pCold) for improved solubility

  • Inducible systems to control expression level

• Expression optimization:

  • Test multiple induction temperatures (16°C, 25°C, 37°C)

  • Vary inducer concentration (0.1-1.0 mM IPTG)

  • Optimize expression duration (4 hours to overnight)

  • Consider auto-induction media for high-density cultures

• Purification strategy:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Size exclusion chromatography for final polishing

  • Ion exchange chromatography as needed

The small size of IF-1 (typically <10 kDa) makes it amenable to various expression strategies, though careful optimization is necessary to ensure proper folding and function.

What functional assays can be employed to verify the activity of recombinant B. quintana IF-1?

Verifying the biological activity of recombinant B. quintana IF-1 requires assays that measure its function in translation initiation. Several complementary approaches can be employed:

• In vitro translation assays:

  • Reconstituted translation system with purified components

  • Measurement of translation efficiency using reporter constructs (luciferase, GFP)

  • Quantification of 30S ribosomal subunit binding

• Complementation studies:

  • Use of E. coli infA conditional mutants

  • Transformation with B. quintana infA expression constructs

  • Assessment of growth restoration under non-permissive conditions

• Structural integrity verification:

  • Circular dichroism to confirm secondary structure

  • Nuclear magnetic resonance for tertiary structure analysis

  • Thermal shift assays to assess protein stability

• Interaction studies:

  • Surface plasmon resonance to measure binding to ribosomal components

  • Microscale thermophoresis for quantitative binding parameters

  • Pull-down assays to identify interaction partners

The essentiality of IF-1 in bacterial cells has been demonstrated in E. coli, where cells depleted of this factor exhibit few polysomes . Similar phenotypes would be expected in complementation experiments using B. quintana infA.

How might fundamental research on B. quintana IF-1 contribute to understanding donor-derived infection in transplantation?

B. quintana infection has been documented as a donor-derived pathogen in solid organ transplantation, representing an emerging public health concern . Research on B. quintana IF-1 could contribute to understanding these infections through several avenues:

• Molecular detection methods:

  • Development of infA-targeted PCR assays for donor screening

  • Implementation in transplant protocols for high-risk donors

• Strain typing and virulence assessment:

  • Sequence variation in infA as potential markers for strain differentiation

  • Correlation between specific infA variants and clinical outcomes

• Pathogenesis understanding:

  • Role of translation regulation in establishing infection in immunosuppressed hosts

  • Adaptation mechanisms during transmission from donor to recipient

Current guidelines for managing donor-derived B. quintana infection stratify risk based on donor characteristics and test results :

Understanding the molecular biology of essential B. quintana components like IF-1 could improve risk assessment and management strategies for transplant recipients.

What insights can comparative analysis of IF-1 provide about B. quintana's evolution in different host species?

B. quintana demonstrates remarkable host adaptability, having been isolated from humans, Japanese macaques, and rhesus macaques. Comparative analysis of highly conserved genes like infA across strains from different hosts can provide evolutionary insights:

• Molecular clock analysis:

  • Assessment of substitution rates in infA compared to other genes

  • Dating divergence events between strains from different hosts

• Selection pressure analysis:

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

  • Identification of specific amino acid residues under selection

• Host adaptation signatures:

  • Comparison of regulatory regions controlling infA expression

  • Assessment of codon usage patterns in different host-adapted strains

How might targeting B. quintana IF-1 function offer novel therapeutic approaches for persistent infections?

Translation initiation factors represent potential therapeutic targets due to their essential nature and structural differences from eukaryotic counterparts. Several research directions could explore IF-1 as a therapeutic target:

• Small molecule inhibitor development:

  • High-throughput screening against recombinant B. quintana IF-1

  • Structure-based drug design targeting IF-1 interaction interfaces

  • Validation in cellular infection models

• Peptide-based inhibitors:

  • Design of peptide mimetics that compete with IF-1 binding sites

  • Cell-penetrating peptide conjugates for intracellular delivery

  • Assessment of specificity against human translation factors

• Antisense strategies:

  • Phosphorodiamidate morpholino oligomers (PMOs) targeting infA mRNA

  • Delivery systems optimized for intracellular bacteria

  • Combinatorial approaches with conventional antibiotics

The challenge of treating B. quintana infections is underscored by their ability to persist in the bloodstream for prolonged periods and cause relapsing fever . Targeting essential cellular processes like translation initiation could provide alternative therapeutic strategies for persistent infections.

What role might IF-1 play in the variable expression of virulence factors during chronic B. quintana infection?

B. quintana demonstrates remarkable adaptability during chronic infection, including variable expression of outer membrane proteins and genomic rearrangements . The potential role of translation factors in this adaptability represents an intriguing research question:

• Translational regulation hypothesis:

  • IF-1 might participate in selective translation of specific mRNAs during stress

  • Altered IF-1 activity could influence the expression of virulence factors

• Methodological approach:

  • Ribosome profiling under different infection conditions

  • Assessment of mRNA association with ribosomes in wild-type vs. IF-1 depleted conditions

  • Correlation with protein expression patterns

• Integration with known adaptation mechanisms:

  • Relationship between translational regulation and the documented variable expression of outer membrane proteins (Vomp)

  • Potential coordination between genomic rearrangements and translational adaptation

Studies have documented that B. quintana undergoes genomic rearrangements during chronic infection, including deletion of Vomp family genes . In one study, progressive genomic rearrangement was observed during infection, with a heterogeneous population at day 12 post-infection becoming more homogeneous by day 70 . Understanding how translational processes might complement these genetic changes could provide a more comprehensive picture of bacterial adaptation during chronic infection.