Recombinant Rickettsia conorii SURF1-like protein (RC1113)

How does RC1113 relate to other SURF1 proteins, and what is its predicted function?

RC1113 belongs to the SURF1 protein family that includes the human SURF1 protein, yeast SHY1, and other rickettsial proteins. While human SURF1 functions as a mitochondrial assembly factor for cytochrome c oxidase , RC1113's function in Rickettsia remains less characterized.

Based on homology studies, RC1113 may be involved in electron transport or cellular respiration processes in Rickettsia conorii. The protein is part of the surfeit gene cluster, although the gene organization differs between rickettsial and mammalian genomes . Unlike human SURF1 mutations that cause Leigh syndrome, the role of RC1113 appears to be specific to rickettsial biology and potentially pathogenesis.

What are the optimal conditions for recombinant expression of RC1113?

Successful expression of recombinant RC1113 has been achieved using E. coli as the expression system. The methodology involves:

• Cloning the full-length RC1113 gene (1-240aa) from Rickettsia conorii genomic DNA

• Incorporating an N-terminal His-tag for purification purposes

• Using an IPTG-inducible promoter system for controlled expression

• Optimizing expression at lower temperatures (typically 16-25°C) to enhance proper folding

Expression vectors such as pET-22b have proven effective for RC1113 expression, similar to methods used for other rickettsial proteins like Sca1, where researchers employed directional restriction endonuclease-mediated insertion .

What purification strategies yield the highest purity and activity for recombinant RC1113?

Purification of His-tagged RC1113 typically involves:

• Cell lysis using sonication or pressure-based methods in Tris/PBS-based buffer

• Initial capture using nickel affinity chromatography

• Removal of contaminating proteins through stringent washing steps

• Elution with imidazole gradient

• Buffer exchange to remove imidazole and establish storage conditions with 6% trehalose, pH 8.0

Final purification to >90% homogeneity can be confirmed using SDS-PAGE. For experimental applications requiring higher purity, additional size exclusion chromatography may be employed.

How should recombinant RC1113 be stored to maintain stability and activity?

Storage recommendations based on experimental data include:

• Short-term storage (up to one week): 4°C in Tris/PBS-based buffer with 6% trehalose at pH 8.0

• Long-term storage: -20°C/-80°C with 50% glycerol as a cryoprotectant

• Reconstitution from lyophilized form: Using deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Aliquoting to avoid repeated freeze-thaw cycles, which significantly decrease protein stability

Single-use aliquots are strongly recommended as repeated freeze-thawing causes degradation and reduced activity.

What are the most effective experimental approaches to study RC1113 function?

Several methodological approaches have proven valuable for studying RC1113 and similar rickettsial proteins:

• Heterologous Expression Systems: Expressing RC1113 in E. coli outer membrane to study membrane localization and function

• Protein-Protein Interaction Studies: Pull-down assays and co-immunoprecipitation to identify interaction partners

• Cellular Localization: Immunofluorescence microscopy using anti-His antibodies or specific anti-RC1113 antibodies

• Functional Assays: Similar to studies on other rickettsial proteins like Sca1, using soluble RC1113 peptides to examine inhibition of bacterial association with host cells

Researchers studying rickettsial surface proteins have successfully used these approaches to determine roles in adhesion, invasion, and host-pathogen interactions .

How can RC1113 be used to investigate Rickettsia-host cell interactions?

To investigate potential roles of RC1113 in host-pathogen interactions:

• Generate recombinant RC1113 fragments fused to GST or other tags for interaction studies

• Develop cell adhesion assays using human endothelial cells (primary targets of Rickettsia)

• Perform inhibition assays with anti-RC1113 antibodies to test if they block infection

• Compare RC1113 expression levels between different growth conditions (human cells vs. tick cells)

Transcriptomic studies have revealed that rickettsial genes show differential expression between human and tick host cells, suggesting RC1113 might be differentially regulated depending on the host environment .

What methodologies can detect RC1113 expression during Rickettsia infection cycles?

Detection of RC1113 expression can be achieved through:

• RT-PCR: Using primers specific to RC1113 mRNA to quantify transcription levels

• Western blotting: Using anti-His antibodies for recombinant protein or custom antibodies against RC1113

• Mass spectrometry: For proteome-wide studies to identify RC1113 expression patterns

• RNA-Seq: To analyze transcriptional regulation of RC1113 during different infection stages

Studies on rickettsial transcriptomes have employed Terminator 5′-phosphate-dependent exonuclease (TEX) treatment to determine transcriptional start sites, which could be applied to RC1113 .

How does RC1113 vary among different Rickettsia conorii subspecies?

Rickettsia conorii has been classified into four subspecies: R. conorii subsp. conorii, R. conorii subsp. indica, R. conorii subsp. israelensis, and R. conorii subsp. caspia . Comparative analysis shows:

These variations may contribute to different pathogenicity patterns observed among R. conorii subspecies. For instance, R. conorii subsp. israelensis is associated with more severe disease manifestations compared to R. conorii subsp. conorii .

What evolutionary insights can be gained from analyzing RC1113 across Rickettsia species?

Evolutionary analysis of SURF1-like proteins across rickettsial species reveals:

• SURF1-like proteins appear conserved across the spotted fever group (SFG) rickettsiae

• Sequence conservation suggests functional importance in rickettsial biology

• Unlike other surface proteins such as Sca1, RC1113 shows fewer signs of selective pressure from host immune responses

• The presence of RC1113 homologs across rickettsial species indicates acquisition prior to speciation events

Comparison with mitochondrial SURF1 proteins can provide insights into the evolutionary relationship between rickettsiae and mitochondria, supporting the endosymbiotic theory of mitochondrial origin.

How can multi-locus sequence typing (MLST) approaches incorporating RC1113 enhance Rickettsia classification?

While standard MLST approaches for Rickettsia typically include genes such as 16S rDNA, gltA, ompA, ompB, and sca4 , inclusion of RC1113 could provide additional resolution:

• RC1113 sequences could be incorporated into existing MLST schemes for finer subspecies differentiation

• Phylogenetic analyses using RC1113 might reveal relationships not apparent with current marker genes

• Specific RC1113 primers can be designed for PCR-based detection and typing assays, similar to approaches used for ompA and ompB genes

A comprehensive MLST approach incorporating RC1113 would provide greater resolution for epidemiological studies of spotted fever rickettsial diseases.

What techniques can be employed to investigate the potential role of RC1113 in rickettsial virulence?

To determine if RC1113 contributes to virulence:

• Comparative Transcriptomics: Analyze RC1113 expression between highly virulent strains (e.g., R. conorii Israeli spotted fever strain) and less virulent strains (e.g., R. massiliae)

• Host Cell Models: Assess effects of recombinant RC1113 on human microvascular endothelial cell (HMEC-1) viability and permeability

• Protein Blocking Studies: Use anti-RC1113 antibodies to block protein function during infection

• Animal Models: Measure infection outcomes in animal models challenged with wild-type versus RC1113-neutralized bacteria

Studies on R. conorii Israeli spotted fever strain have shown its ability to cause significant endothelial cell injury and increased permeability compared to less virulent rickettsial species, potentially indicating differences in surface protein functions .

How might RC1113 interact with tick host cells in comparison to human cells?

Understanding RC1113's dual roles in tick vectors versus human hosts represents an advanced research area:

• Comparative transcriptomics of RC1113 during growth in tick cells versus human endothelial cells can reveal differential regulation

• Functional assays examining RC1113's role in tick cell entry versus human cell entry

• Investigation of RC1113's potential role in transovarial/transstadial transmission within tick vectors

• Analysis of how RC1113 might contribute to rickettsial survival during tick starvation periods

Research on Rickettsia rickettsii has shown that rickettsial infection can increase tick survival during starvation . Similar studies examining RC1113 expression during tick starvation could reveal whether this protein contributes to rickettsial persistence in vectors.

What are the common challenges in working with recombinant RC1113, and how can they be addressed?

Researchers working with recombinant RC1113 encounter several technical challenges:

• Protein Solubility Issues: RC1113 contains hydrophobic domains that can cause aggregation

  • Solution: Express as fusion proteins with solubility tags like GST or MBP

  • Use lower induction temperatures (16-20°C) and reduced IPTG concentrations

• Protein Folding: Ensuring proper folding of membrane-associated proteins

  • Solution: Consider cell-free expression systems or specialized E. coli strains designed for membrane protein expression

• Functional Validation: Confirming biological activity of the recombinant protein

  • Solution: Develop functional assays based on predicted roles in adhesion or membrane function

  • Compare results from different expression constructs with varying tags and fusion partners

• Cross-Reactivity in Immunological Detection: Antibodies may cross-react with homologous proteins

  • Solution: Develop highly specific monoclonal antibodies targeting unique RC1113 epitopes

  • Validate antibody specificity against other SURF1-like proteins

What quality control measures should be implemented for RC1113 experimental studies?

To ensure experimental reproducibility with RC1113:

• Purity Assessment: Confirm >90% purity by SDS-PAGE and mass spectrometry

• Activity Verification: Develop functional assays relevant to predicted protein function

• Endotoxin Testing: Ensure preparations are endotoxin-free for cell-based assays

• Sequence Verification: Confirm protein identity by peptide mass fingerprinting or N-terminal sequencing

• Stability Testing: Monitor protein stability under experimental conditions using dynamic light scattering or thermal shift assays

These quality control measures are essential for generating reliable and reproducible research data with recombinant RC1113 .