Recombinant Rickettsia bellii SURF1-like protein (RBE_0359)

What is Rickettsia bellii SURF1-like protein (RBE_0359)?

Rickettsia bellii SURF1-like protein (RBE_0359) is a full-length protein (241 amino acids) from Rickettsia bellii, a species that is not associated with human disease. The protein is identified by UniProt ID Q1RJM4 and shares structural similarities with SURF1 proteins, which are typically involved in cellular respiration processes. The complete amino acid sequence is: MKTKLTVLITFIILVLLGFWQLNRLKEKKLFLASMQENLTSPAIDLAKIQDNLPYHKVKI TGHFLPDKDIYLYGRRSMSSEKDGYYLVTPFKTDEDKIILVARGWFSNRNKNIITQATND QPHELIGVTMPSEKTRSYLPANDIKNNVWLTLDLQEASKVLGLNLENFYLIEESKDISNL DILLPLSINHLAAIRNDHLEYAFTWFGLAASLVVIYRIYKRSVSSRGLETRSRIKQDKSS F .

How does Rickettsia bellii differ biologically from pathogenic Rickettsia species?

Rickettsia bellii exhibits significant biological differences from pathogenic Rickettsia species. While R. bellii can invade both endothelial cells and macrophage-like cells similar to pathogenic species, it can only proliferate within endothelial cells. In contrast, pathogenic Rickettsia species such as R. rickettsii and R. parkeri can successfully proliferate in both endothelial cells and macrophages . This differential growth characteristic represents a key distinction that likely contributes to R. bellii's non-pathogenic nature in humans. Additionally, R. bellii shows significant co-localization with lysosomal markers in macrophages, suggesting an inability to escape lysosomal degradation, which pathogenic species can effectively avoid .

What are the proper storage and reconstitution conditions for recombinant RBE_0359?

For optimal storage of recombinant RBE_0359 protein:

• Store at -20°C/-80°C upon receipt

• Aliquot the protein to avoid repeated freeze-thaw cycles

• The protein is supplied as a lyophilized powder in Tris/PBS-based buffer with 6% Trehalose at pH 8.0

• For reconstitution, briefly centrifuge the vial before opening

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being standard) for long-term storage

• Working aliquots can be stored at 4°C for up to one week

How should experiments be designed to compare RBE_0359 with homologous proteins from pathogenic Rickettsia species?

When designing comparative experiments between RBE_0359 and homologous proteins from pathogenic Rickettsia species, researchers should implement a multi-faceted approach:

• Cell type selection: Include both endothelial cells (e.g., EA.hy926) and macrophage-like cells (e.g., PMA-differentiated THP-1) to observe differential growth patterns between R. bellii and pathogenic species .

• Time course analysis: Establish multiple time points (e.g., 0h, 24h, 48h, 72h) to capture growth kinetics, as R. bellii shows distinctly different proliferation patterns compared to pathogenic species .

• Quantification methods: Employ multiple quantitative techniques:

  • qPCR to determine the ratio of rickettsial DNA (e.g., sca1 gene) to host cell DNA (e.g., actin gene)

  • Fluorescence microscopy-based growth assays

  • Immunofluorescence confocal microscopy for protein localization studies

• Controls: Include pathogenic species (R. rickettsii, R. parkeri) and avirulent strains (e.g., R. rickettsii strain Iowa) as comparative controls .

• Variables and controls: Following proper experimental design principles:

  • Independent variables: Rickettsia species, cell type, time point

  • Dependent variables: Growth rate, cellular localization, co-localization with cellular markers

  • Control variables: MOI (multiplicity of infection), culture conditions, cell passage number

What methodological approaches can effectively detect cellular localization of RBE_0359?

To determine the cellular localization of RBE_0359, researchers should employ a combination of complementary techniques:

• Immunofluorescence confocal microscopy:

  • Use specific antibodies against RBE_0359 with fluorescent tags

  • Co-stain with cellular compartment markers such as LAMP-2 (lysosomal marker) and Cathepsin D (mature lysosomal marker)

  • Analyze z-stack images to determine three-dimensional localization

  • Generate RGB profile plots to document relative fluorescence intensity and determine co-localization events

• Subcellular fractionation:

  • Separate cellular components through differential centrifugation

  • Analyze fractions using Western blot with anti-RBE_0359 antibodies

  • Compare distribution patterns with known markers for different cellular compartments

• Live-cell imaging:

  • Create fluorescently tagged RBE_0359 constructs

  • Monitor trafficking and localization in real-time

  • Compare trafficking patterns with similar constructs from pathogenic species

• Electron microscopy:

  • Utilize immunogold labeling for precise subcellular localization

  • Achieve nanometer-scale resolution to determine exact positioning relative to cellular structures

How do researchers accurately quantify growth differences between R. bellii and pathogenic Rickettsia species?

For accurate quantification of growth differences between R. bellii and pathogenic Rickettsia species, researchers should implement multiple complementary methods:

• Quantitative PCR (qPCR):

  • Extract total genomic DNA from infected cells at defined time points

  • Amplify both rickettsial genes (e.g., sca1) and host cell genes (e.g., actin)

  • Calculate the ratio of rickettsial to host cell DNA as a measure of bacterial load

  • Analyze changes in this ratio over time to determine growth kinetics

• Fluorescence microscopy-based growth assays:

  • Fix cells at different time points post-infection

  • Stain with fluorescently labeled antibodies specific to Rickettsia

  • Count the number of bacteria per cell across multiple fields

  • Calculate average bacterial load per cell over time

• Plaque assays or focus-forming unit assays:

  • Harvest bacteria from infected cells at different time points

  • Perform serial dilutions and infect fresh cell monolayers

  • Count plaques or foci after appropriate incubation

  • Calculate the increase in viable bacteria over time

What factors explain the different growth characteristics of R. bellii compared to pathogenic Rickettsia species in macrophages?

Several mechanisms likely contribute to the differential growth characteristics of R. bellii compared to pathogenic Rickettsia species in macrophages:

• Lysosomal escape mechanisms:

  • Pathogenic Rickettsia species (R. rickettsii, R. parkeri) show minimal co-localization with lysosomal markers

  • R. bellii shows significant co-localization with both Cathepsin D and LAMP-2

  • This indicates pathogenic species possess mechanisms to escape lysosomal degradation that R. bellii lacks

• Phagosomal maturation interference:

  • Pathogenic Rickettsia may secrete effector proteins that inhibit phagolysosomal fusion

  • R. bellii likely lacks these effectors or expresses them at insufficient levels

  • This difference results in R. bellii being trafficked to mature lysosomes and subsequently degraded

• Membrane composition differences:

  • Variations in outer membrane proteins may affect interactions with host cell membranes

  • Different surface protein modifications might alter recognition by host cell defense mechanisms

  • Pathogenic species may have evolved specific membrane adaptations for macrophage survival

• Evolutionary divergence:

  • R. bellii represents one of the earliest diverging lineages of Rickettsia

  • The ability to proliferate within macrophages may be a derived trait in pathogenic lineages

  • This suggests macrophage survival is a key evolutionary step in the development of pathogenicity

How can researchers experimentally determine if RBE_0359 contributes to the inability of R. bellii to escape lysosomal degradation?

To determine if RBE_0359 contributes to R. bellii's inability to escape lysosomal degradation, researchers should consider the following experimental approaches:

• Gene knockout or knockdown studies:

  • Create RBE_0359 deletion mutants or use RNA interference approaches

  • Assess whether mutant R. bellii shows altered co-localization with lysosomal markers

  • Determine if deletion affects survival in macrophages

  • Compare growth kinetics of wild-type versus mutant strains in different cell types

• Heterologous expression experiments:

  • Express RBE_0359 in pathogenic Rickettsia species

  • Express homologous proteins from pathogenic species in R. bellii

  • Assess whether these exchanges affect lysosomal escape capabilities

  • Quantify changes in co-localization with lysosomal markers (Cathepsin D, LAMP-2)

• Domain swapping experiments:

  • Create chimeric proteins between RBE_0359 and homologs from pathogenic species

  • Express these chimeras in appropriate Rickettsia strains

  • Identify domains responsible for differences in lysosomal escape capabilities

• Co-localization time course studies:

  • Track the intracellular fate of R. bellii at multiple time points post-infection

  • Compare with pathogenic species to identify when trafficking pathways diverge

  • Use multiple lysosomal and endosomal markers to determine precise trafficking routes

What advanced microscopy techniques are most effective for analyzing RBE_0359 interactions with host cell components?

For analyzing RBE_0359 interactions with host cell components, several advanced microscopy techniques offer unique advantages:

• Super-resolution microscopy:

  • Techniques such as STORM, PALM, or STED provide resolution beyond the diffraction limit

  • Can resolve structures at 20-50 nm resolution compared to ~250 nm in conventional microscopy

  • Enables precise visualization of protein clustering and nanoscale distribution

  • Particularly valuable for examining membrane-associated proteins like RBE_0359

• Live-cell confocal microscopy with FRET:

  • Fluorescence Resonance Energy Transfer (FRET) detects molecular proximity (<10 nm)

  • Can confirm direct interactions between RBE_0359 and host proteins

  • Time-lapse imaging reveals dynamic interaction patterns during infection

• Correlative Light and Electron Microscopy (CLEM):

  • Combines fluorescence microscopy with electron microscopy

  • First identify proteins of interest using fluorescence

  • Then examine the same regions with electron microscopy for ultrastructural context

  • Provides both molecular specificity and high-resolution structural information

• Lattice light-sheet microscopy:

  • Enables long-term 3D imaging of living cells with minimal phototoxicity

  • Ideal for tracking RBE_0359 trafficking in real-time

  • Can capture rapid dynamic events during bacterial invasion and intracellular movement

• Expansion microscopy:

  • Physically expands biological specimens while maintaining relative spatial relationships

  • Achieves super-resolution imaging with conventional microscopes

  • Particularly useful for crowded intracellular environments

What are the optimal conditions for expressing and purifying recombinant RBE_0359 protein?

For optimal expression and purification of recombinant RBE_0359 protein:

• Expression system:

  • Use E. coli strains optimized for membrane protein expression (e.g., C41(DE3), C43(DE3))

  • Consider codon-optimized synthetic gene to improve expression efficiency

  • Select expression vectors with appropriate promoters (T7) and fusion tags (His-tag)

• Culture conditions:

  • Grow initial culture at 37°C to OD600 of 0.6-0.8

  • Reduce temperature to 16-25°C before induction to enhance proper folding

  • Induce with lower IPTG concentrations (0.1-0.5 mM) to prevent inclusion body formation

  • Extended induction times (16-24 hours) at lower temperatures often improve yield

• Harvest and lysis:

  • Harvest cells by centrifugation (5000 x g, 10 min, 4°C)

  • Resuspend in buffer containing protease inhibitors

  • Use gentle lysis methods (lysozyme treatment followed by sonication)

  • Separate soluble and insoluble fractions by centrifugation

• Purification strategy:

  • Immobilized metal affinity chromatography (IMAC) using the His-tag

  • Consider additional purification steps (ion exchange, size exclusion)

  • Evaluate protein purity by SDS-PAGE (aim for >90% purity)

  • Use Tris/PBS-based buffer with 6% Trehalose at pH 8.0 for final storage

What analytical methods are most effective for verifying the structural integrity of purified RBE_0359?

To verify the structural integrity of purified RBE_0359, researchers should employ multiple complementary analytical methods:

• Purity assessment:

  • SDS-PAGE with Coomassie or silver staining (aim for >90% purity)

  • Western blot using anti-His antibodies or specific anti-RBE_0359 antibodies

  • Mass spectrometry to confirm protein identity and detect contaminants

  • High-performance liquid chromatography (HPLC) to assess homogeneity

• Structural characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure elements

  • Fluorescence spectroscopy to examine tertiary structure and folding

  • Dynamic light scattering (DLS) to detect aggregation and assess size distribution

  • Limited proteolysis to identify stable domains and proper folding

  • Thermal shift assays to evaluate protein stability

• Functional validation:

  • Binding assays with potential interaction partners

  • Liposome association assays to verify membrane protein characteristics

  • Comparing properties with computationally predicted structural features

• Advanced structural analysis:

  • Small-angle X-ray scattering (SAXS) for low-resolution structural information

  • Cryo-electron microscopy for membrane proteins resistant to crystallization

  • Nuclear magnetic resonance (NMR) for dynamic structural information

How can researchers design experiments to identify potential interaction partners of RBE_0359?

To identify potential interaction partners of RBE_0359, researchers should consider multiple complementary approaches:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express RBE_0359 with an affinity tag (His-tag already present)

  • Perform pull-down experiments from cell lysates

  • Identify co-purifying proteins by mass spectrometry

  • Compare results with control pull-downs to identify specific interactions

• Proximity-based labeling:

  • Create fusion proteins of RBE_0359 with BioID or APEX2

  • Express in relevant cell types or in R. bellii

  • Biotinylate proteins in close proximity to RBE_0359

  • Purify biotinylated proteins and identify by mass spectrometry

• Yeast two-hybrid screening:

  • Use RBE_0359 as bait against host cell or bacterial prey libraries

  • Screen for positive interactions

  • Validate initial hits with secondary assays

  • Consider membrane yeast two-hybrid for this membrane protein

• Co-immunoprecipitation with specific antibodies:

  • Generate specific antibodies against RBE_0359

  • Perform immunoprecipitation from infected cells

  • Identify co-precipitating proteins by mass spectrometry

  • Validate interactions with reciprocal co-immunoprecipitation

• Crosslinking mass spectrometry:

  • Use chemical crosslinkers to stabilize transient interactions

  • Digest crosslinked complexes and analyze by specialized mass spectrometry

  • Identify crosslinked peptides to map interaction interfaces

  • Provides both identification of partners and structural information

How does the amino acid sequence of RBE_0359 compare with homologous proteins in pathogenic Rickettsia species?

A comparative analysis of RBE_0359 with homologous proteins in pathogenic Rickettsia species would involve:

• Sequence alignment analysis:

  • Align RBE_0359 sequence with homologs from pathogenic species (R. rickettsii, R. parkeri, etc.)

  • Identify conserved regions likely important for core protein function

  • Highlight species-specific variations that might correlate with pathogenicity

  • Calculate sequence identity and similarity percentages

• Domain structure comparison:

  • Identify functional domains within RBE_0359 and homologous proteins

  • Determine if pathogenic species contain additional domains or motifs

  • Look for sequence insertions/deletions that might alter function

  • Analyze transmembrane regions and topology predictions

• Evolutionary analysis:

  • Construct phylogenetic trees to visualize evolutionary relationships

  • Calculate evolutionary rates for different protein regions

  • Identify positions under positive selection in pathogenic lineages

  • Correlate sequence changes with the acquisition of virulence traits

• Structural prediction comparison:

  • Generate structural models of RBE_0359 and homologs

  • Compare predicted secondary and tertiary structures

  • Identify structural differences that might affect protein function

  • Focus on regions that interact with host cell components

What experimental techniques are most effective for analyzing functional differences between RBE_0359 and homologs from pathogenic species?

To analyze functional differences between RBE_0359 and homologs from pathogenic species, researchers should employ:

• Heterologous expression systems:

  • Express RBE_0359 and homologs in the same cellular background

  • Compare localization patterns, stability, and interacting partners

  • Assess functional complementation capabilities

  • Determine if expression affects cellular processes differently

• Chimeric protein analysis:

  • Create domain swap constructs between RBE_0359 and pathogenic homologs

  • Express in appropriate cell types or bacterial systems

  • Identify which domains or regions are responsible for functional differences

  • Map specific regions responsible for differential lysosomal escape capabilities

• Infection models with protein knockdown/complementation:

  • Create knockdown or knockout strains for RBE_0359 and homologs

  • Complement with various versions of the proteins

  • Assess changes in infection dynamics, intracellular trafficking, and survival

  • Determine if RBE_0359 expression affects the behavior of pathogenic species

• Host cell response analysis:

  • Compare host cell transcriptional and proteomic responses to RBE_0359 versus homologs

  • Identify differentially regulated pathways and processes

  • Determine if pathogenic homologs trigger specific host response patterns

  • Correlate molecular signatures with infection outcomes

How might studying RBE_0359 contribute to developing intervention strategies against pathogenic Rickettsia?

Studying RBE_0359 could contribute to intervention strategies against pathogenic Rickettsia through multiple approaches:

• Vaccine development:

  • Non-pathogenic R. bellii could potentially serve as a live attenuated vaccine platform

  • Identifying differences between RBE_0359 and homologs in pathogenic species could guide subunit vaccine design

  • Engineering chimeric proteins that maintain immunogenicity but lack virulence determinants

  • Using understanding of differential intracellular trafficking to create strategically attenuated strains

• Therapeutic target identification:

  • Determining how pathogenic homologs enable lysosomal escape may reveal novel drug targets

  • Identifying protein regions unique to pathogenic species could allow selective targeting

  • Developing inhibitors that specifically block functions essential for intracellular survival

  • Creating peptide-based inhibitors derived from RBE_0359 sequences that might interfere with pathogenic homolog function

• Diagnostic development:

  • Creating assays that distinguish between pathogenic and non-pathogenic Rickettsia based on protein differences

  • Developing antibodies that specifically recognize pathogenic variants

  • Designing nucleic acid tests targeting sequence differences between RBE_0359 and pathogenic homologs

  • Improving rapid diagnostic capabilities for rickettsial infections

• Pathogenesis understanding:

  • Using R. bellii as a comparative model to identify core virulence determinants

  • Understanding the molecular basis for macrophage survival in pathogenic species

  • Elucidating the evolutionary trajectory from non-pathogenic to pathogenic Rickettsia

  • Identifying host factors that restrict R. bellii but not pathogenic species

What are the most significant research gaps in our understanding of RBE_0359?

Despite growing knowledge about RBE_0359, several significant research gaps remain:

• Structural characterization:

  • The three-dimensional structure of RBE_0359 remains undetermined

  • Structural comparison with homologs from pathogenic species is lacking

  • Structure-function relationships for specific protein domains are poorly understood

• Precise biological function:

  • The exact cellular function of RBE_0359 in R. bellii remains unclear

  • Whether it contributes directly to the inability to proliferate in macrophages is undetermined

  • Its role in normal R. bellii physiology needs further investigation

• Host interaction networks:

  • Comprehensive identification of host cell proteins that interact with RBE_0359 is missing

  • Differences in interaction profiles between RBE_0359 and pathogenic homologs remain largely unexplored

  • The functional consequences of these differential interactions require further study

• Evolutionary history:

  • The evolutionary trajectory of SURF1-like proteins in Rickettsia species is not fully mapped

  • The selective pressures that shaped divergence between pathogenic and non-pathogenic forms are poorly understood

  • The timing of functional divergence relative to the acquisition of pathogenicity remains unclear

How might future research on RBE_0359 advance our understanding of bacterial pathogenesis more broadly?

Future research on RBE_0359 could provide broader insights into bacterial pathogenesis through:

• Understanding pathogen evolution:

  • Comparing non-pathogenic and pathogenic Rickettsia species offers a window into how pathogens evolve

  • Identifying the minimal genetic changes needed to convert a non-pathogen to a pathogen

  • Elucidating how new virulence mechanisms emerge and are selected for during evolution

• Illuminating host-pathogen interfaces:

  • Determining how subtle protein differences dictate drastically different infection outcomes

  • Understanding how bacteria evade or manipulate host defense mechanisms

  • Identifying conserved host pathways targeted by diverse intracellular pathogens

• Advancing therapeutic strategies:

  • Developing new approaches to combat intracellular pathogens

  • Creating broadly applicable strategies for interfering with bacterial protein functions

  • Designing novel vaccination approaches based on non-pathogenic variants

• Improving experimental systems:

  • Establishing R. bellii as a safer model system for studying intracellular bacterial biology

  • Developing new tools for genetic manipulation of challenging bacterial systems

  • Creating innovative approaches for studying membrane protein functions in intracellular bacteria