Recombinant Rickettsia massiliae Translation initiation factor IF-2 (infB), partial

What is Rickettsia massiliae and how does it relate to other rickettsial species?

Rickettsia massiliae is a tick-borne obligate intracellular alpha-proteobacteria belonging to the spotted fever group (SFG) of Rickettsia. It causes spotted fever in humans and was first isolated in 1992. R. massiliae possesses a 1.3-Mb circular chromosome and a 15.3-kb plasmid . Phylogenetically, R. massiliae clusters with other SFG rickettsiae but contains unique genomic elements, including 14 tra genes presumably involved in pilus formation and conjugal DNA transfer, which were acquired through lateral gene transfer from a species related to Rickettsia bellii .

Molecular detection of R. massiliae is primarily accomplished through PCR targeting genes encoding citrate synthase (gltA) and outer membrane protein (ompA), followed by sequence and phylogenetic analysis . R. massiliae has been detected in multiple Rhipicephalus tick species globally, including R. turanicus, R. sanguineus s.l., and Ixodes ricinus .

What is the function of bacterial translation initiation factor IF-2 (infB)?

Translation initiation factor IF-2 plays a critical role in bacterial protein synthesis by:

• Binding to the 30S ribosomal subunit and facilitating the recruitment of the initiator tRNA (fMet-tRNA)

• Promoting the formation of the 30S initiation complex

• Supporting the joining of the 50S ribosomal subunit to form the 70S initiation complex

• Hydrolyzing GTP during these processes, which provides energy for conformational changes

In obligate intracellular bacteria like R. massiliae, translation factors are particularly important for adaptation to the host cell environment. Research has shown that Rickettsia species can affect host translation machinery, with significant reductions observed in the abundance of translation initiation factor eIF-2B subunit delta in infected host cells . This suggests a complex interplay between bacterial and host translation systems during infection.

Why is the study of partial recombinant IF-2 important in rickettsial research?

Studying partial recombinant IF-2 from R. massiliae is valuable for several reasons:

• Diagnostic development: Recombinant rickettsial proteins are crucial for developing serological assays. Traditional IFA methods require specialized BSL-3 facilities for antigen preparation, whereas recombinant protein production only requires BSL-2 facilities .

• Vaccine candidates: Surface proteins from Rickettsia are subject to intense positive natural selection, making them potential vaccine targets .

• Evolutionary insights: Translation factors like IF-2 can provide phylogenetic information about the evolutionary relationships between rickettsial species .

• Functional studies: Partial recombinant proteins allow for domain-specific investigations of protein function without the challenges of expressing the entire protein.

What expression systems are most effective for producing recombinant rickettsial proteins?

For successful expression of recombinant R. massiliae IF-2 (infB), several expression systems can be employed, each with advantages and limitations:

For R. massiliae IF-2, E. coli expression using the pSY5 plasmid has been successfully employed for other rickettsial proteins . When expressing partial IF-2, it's essential to ensure the selected region contains complete functional domains to maintain biological activity.

What purification and validation protocols are recommended for recombinant IF-2?

A robust purification and validation workflow for recombinant R. massiliae IF-2 includes:

• Initial purification:

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

  • Glutathione affinity chromatography for GST-tagged proteins

  • Size exclusion chromatography for final polishing

• Quality assessment:

  • SDS-PAGE to confirm molecular weight and purity

  • Western blot using anti-His/GST antibodies and/or R. massiliae-specific antisera

  • Mass spectrometry for protein identification and detection of post-translational modifications

• Functional validation:

  • GTP binding and hydrolysis assays

  • 30S ribosomal subunit binding assays

  • In vitro translation efficiency tests comparing wild-type and mutant versions

• Structural integrity:

  • Circular dichroism spectroscopy

  • Thermal shift assays

  • Limited proteolysis to assess domain folding

When validating recombinant IF-2 activity, it's important to include appropriate controls, including commercially available E. coli IF-2 and other bacterial IF-2 proteins for comparative analysis.

How can we design effective experimental controls when working with recombinant rickettsial proteins?

Robust experimental design for studying R. massiliae IF-2 requires careful consideration of controls:

• Negative controls:

  • Empty vector-transformed E. coli lysates processed identically to the recombinant protein

  • Irrelevant proteins of similar size and charge characteristics

  • Heat-denatured recombinant IF-2 protein

• Positive controls:

  • Commercially available IF-2 from model organisms (E. coli, B. subtilis)

  • Previously characterized recombinant proteins using the same expression system

  • Native IF-2 from related Rickettsia species (if available)

• Internal controls:

  • Include known concentrations of standard proteins for quantification

  • Use multiple detection methods (e.g., fluorescence and colorimetric assays)

• Multi-factorial experimental design:

  • Implement 2×2 factorial designs to evaluate interaction effects between variables

  • Consider fractional factorial designs when resource constraints limit testing all combinations

For example, when testing the functional activity of IF-2, a complete 2×2 factorial design might examine both temperature (25°C vs. 37°C) and pH (6.5 vs. 7.5) to identify optimal conditions and potential interaction effects between these variables.

How can recombinant IF-2 be used to understand host-pathogen interactions during R. massiliae infection?

Recombinant IF-2 can serve as a powerful tool for investigating host-pathogen interactions:

• Interactome studies:

  • Pull-down assays with recombinant IF-2 can identify host cellular factors that interact with bacterial translation machinery

  • Yeast two-hybrid or mammalian two-hybrid screening to identify novel protein-protein interactions

  • Proximity labeling methods (BioID, APEX) in cellular models to identify proteins in close proximity to IF-2 during infection

• Host response investigation:

  • Stimulation of host cells with recombinant IF-2 to measure inflammatory responses

  • Analysis of host translation factor modulation through quantitative proteomics

  • Examination of how R. massiliae IF-2 affects host protein synthesis rates

• Comparative analysis with other Rickettsia species:

  • Different Rickettsia species trigger species-specific proteome signatures in macrophages

  • Recombinant IF-2 from multiple species can be used to identify specific modulatory effects

Recent studies have demonstrated that R. massiliae infection of THP-1 macrophages results in significant effects on host type I interferon responses, mRNA splicing, and protein translation machinery . This suggests that bacterial translation factors may play roles beyond protein synthesis during infection.

What is known about the evolutionary aspects of the infB gene in Rickettsia species?

The evolutionary analysis of the infB gene provides insights into rickettsial evolution:

The phylogeny of Rickettsia using different evolutionary signatures shows that while extensive incongruence between individual gene trees exists, this is more plausibly attributed to systematic error than to horizontal gene transfer in core genes like infB .

What are the challenges and solutions in structural studies of rickettsial IF-2?

Structural characterization of R. massiliae IF-2 presents several challenges:

Recent advances that can overcome these challenges include:

• Hybrid structural approaches:

  • Combining X-ray crystallography of domains with cryo-EM of larger assemblies

  • Integrating small-angle X-ray scattering (SAXS) with NMR spectroscopy

  • Using AlphaFold2 or similar AI tools to predict structures and guide experimental design

• Dynamic structural analysis:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map conformational changes

  • Single-molecule FRET to observe real-time conformational dynamics

  • Time-resolved cryo-EM to capture different functional states

Understanding the structure of R. massiliae IF-2 would provide valuable insights into both bacterial translation mechanisms and potential targets for therapeutic intervention.

How can recombinant IF-2 contribute to improved diagnostics for R. massiliae infections?

Recombinant IF-2 offers promising applications for R. massiliae diagnostics:

• Serological assay development:

  • ELISA-based detection using recombinant IF-2 as a capture antigen

  • Multiplex bead-based assays incorporating multiple rickettsial recombinant proteins

  • Lateral flow immunoassays for point-of-care testing

• Advantages over traditional methods:

  • IFA for rickettsial diseases requires personnel expertise, specialized facilities, and has subjective results interpretation

  • Recombinant protein ELISA only requires BSL-2 facilities and is better suited for high-throughput work in low-income settings

  • Recombinant proteins offer more standardized antigen preparation

• Performance considerations:

  • When developing recombinant protein diagnostics, sensitivity and specificity vary significantly based on the protein used

  • In one comparative study of rickettsial recombinant protein diagnostics, OmpB-typhi demonstrated sensitivity of 72.2% and specificity of 73.4%, while OmpA-L showed poorer performance (61.5% sensitivity, 61.1% specificity)

  • IF-2 diagnostics would need validation against these established recombinant protein targets

For optimal diagnostic performance, combining multiple recombinant antigens (including IF-2) may provide improved sensitivity and specificity compared to single-antigen approaches.

What is the potential for IF-2 as a therapeutic target in rickettsial infections?

Translation initiation factor IF-2 represents a potential therapeutic target due to several characteristics:

• Target validation considerations:

  • Essential role in bacterial protein synthesis

  • Structural and functional differences from mammalian counterparts

  • Accessibility to small molecule inhibitors

• Therapeutic approaches:

  • Small molecule inhibitors of GTPase activity

  • Peptide mimetics disrupting IF-2 interactions with ribosomal components

  • Antisense oligonucleotides targeting infB mRNA

• Challenges in development:

  • Delivery of therapeutics to intracellular bacteria

  • Potential for cross-reactivity with human translation factors

  • Need for penetration of both bacterial and host cell membranes

• Combination therapy potential:

  • IF-2 inhibitors could be synergistic with current antibiotic treatments

  • Targeting bacterial translation could reduce virulence factor production

Current antibiotic treatments for R. massiliae infections typically involve doxycycline, but the development of translation-targeting therapeutics could provide alternatives for resistant cases or patients with contraindications to standard treatments.

What emerging technologies could advance the study of rickettsial translation factors?

Several cutting-edge technologies show promise for advancing R. massiliae IF-2 research:

• CRISPR-based technologies:

  • CRISPR interference (CRISPRi) to modulate host factors that interact with bacterial IF-2

  • CRISPRa to upregulate host defensive factors against rickettsial infection

  • Base editing to introduce specific mutations in host factors that interact with IF-2

• Advanced imaging approaches:

  • Super-resolution microscopy to visualize IF-2 localization during infection

  • Expansion microscopy to enhance visualization of host-pathogen interfaces

  • Live-cell imaging with tagged IF-2 to track dynamics during infection

• Multi-omics integration:

  • Combining transcriptomics, proteomics, and metabolomics to understand the impact of IF-2 on host cellular processes

  • Single-cell approaches to characterize heterogeneity in host responses

  • Spatial transcriptomics to map infection progression in tissues

• Microfluidic systems:

  • Organ-on-chip models to study rickettsial infections in complex tissue environments

  • Droplet-based systems for high-throughput screening of translation inhibitors

  • Microfluidic co-culture systems to model vector-host-pathogen interactions

The integration of these technologies with traditional biochemical approaches will provide unprecedented insights into rickettsial translation factors and their roles in pathogenesis.

How can comparative studies of IF-2 across Rickettsia species inform our understanding of pathogenicity?

Comparative analysis of IF-2 across different Rickettsia species can reveal insights into virulence mechanisms:

• Correlation with disease severity:

  • Compare IF-2 sequence and activity between highly pathogenic species (R. rickettsii) and mildly pathogenic ones (R. massiliae)

  • Identify specific domains or residues that correlate with clinical outcomes

• Host adaptation signatures:

  • Analyze IF-2 from species with different host ranges (human-specific vs. broader host range)

  • Identify potential host-specific adaptations in IF-2 function

• Vector-specific adaptations:

  • Compare IF-2 from Rickettsia species transmitted by different tick vectors

  • Investigate whether IF-2 variations contribute to vector competence

• Experimental approaches:

  • Domain-swapping experiments between IF-2 from different species

  • Site-directed mutagenesis of specific residues identified in comparative analyses

  • Cross-species complementation studies to assess functional conservation

Studies of spotted fever group Rickettsia have demonstrated that they trigger species-specific alterations in host cell responses . Understanding the role of IF-2 in these differential responses could provide valuable insights into pathogenicity mechanisms.