Recombinant Bartonella henselae 30S ribosomal protein S17 (rpsQ)

What is Bartonella henselae and why is the 30S ribosomal protein S17 significant in its study?

Bartonella henselae is a worldwide fastidious bacterium with a feline reservoir that is pathogenic for humans . The 30S ribosomal protein S17 (rpsQ) is one of the crucial components of the bacterial ribosome involved in protein synthesis. This protein has been identified as an important target for understanding B. henselae's molecular biology, phylogenetic relationships, and as a potential target for diagnostic methods.

The significance of studying rpsQ lies in its conservation across Bartonella species while maintaining sufficient sequence variation to be useful in strain typing and phylogenetic analyses. Recent research suggests that ribosomal proteins can be valuable targets for developing molecular diagnostic tools and understanding evolutionary relationships among bacterial strains.

What genomic databases are most reliable for retrieving B. henselae S17 (rpsQ) sequences?

For researchers seeking high-quality B. henselae genomic data, including rpsQ sequences, the BV-BRC (formerly PatricBRC) database has been documented as a reliable resource . Additional recommended databases include:

When retrieving sequences, researchers should consider strain variation, as different B. henselae isolates may show variation in their rpsQ sequences, which can be important for identification and typing purposes .

How does the rpsQ gene from B. henselae compare with other Bartonella species?

Comparative studies of rpsQ sequences across Bartonella species reveal both conserved and variable regions that can be exploited for species-specific identification. While the core functional domains of rpsQ show high conservation due to their essential role in ribosome assembly and function, certain regions exhibit interspecies variation.

Current research suggests that while rpsQ hasn't been widely used as a single target for Bartonella typing, it could potentially be incorporated into multilocus sequence typing (MLST) schemes, which have been shown to be highly effective for Bartonella species differentiation . The existing MLST methods incorporating nine genes have successfully distinguished seven genotypes among human and cat isolates of B. henselae .

What are the most effective PCR protocols for amplifying the B. henselae rpsQ gene?

While the search results don't specifically outline PCR protocols targeting rpsQ in B. henselae, they provide insights into effective molecular approaches for Bartonella species detection that could be adapted for rpsQ amplification:

• Conventional PCR approach:

  • Based on established Bartonella detection methods, researchers could design primers targeting conserved regions flanking the rpsQ gene

  • PCR conditions typically involve initial denaturation at 95°C for 5 minutes, followed by 35-40 cycles of denaturation (95°C, 30 seconds), annealing (temperature optimized for primers, typically 55-60°C, 30 seconds), and extension (72°C, 30-60 seconds)

• LAMP (Loop-Mediated Isothermal Amplification) approach:

  • This method has shown high sensitivity for Bartonella detection (125 fg/reaction for B. quintana)

  • LAMP can be performed isothermally (63°C) within 18 minutes, making it suitable for rapid detection

  • For rpsQ detection, appropriate primers would need to be designed following LAMP primer design principles

• qPCR approach:

  • Real-time PCR has been effectively used for Bartonella detection, though with potentially lower sensitivity than LAMP in some contexts

  • A typical qPCR reaction would include appropriate primers, probe, and master mix in a 20-μL reaction volume

The choice of method should be based on research objectives, available equipment, and required sensitivity/specificity levels.

How can recombinant B. henselae rpsQ protein be efficiently expressed and purified?

For researchers working on expression and purification of recombinant B. henselae rpsQ, a methodological approach based on standard recombinant protein techniques would include:

• Cloning strategy:

  • Amplify the rpsQ gene using primers with appropriate restriction sites

  • Clone into an expression vector (commonly pET series for bacterial expression)

  • Transform into an E. coli expression strain (BL21(DE3) or its derivatives)

• Expression optimization:

  • Test different induction conditions (IPTG concentration, temperature, duration)

  • A typical starting protocol would be 0.5-1.0 mM IPTG at 37°C for 4 hours or 0.2-0.5 mM IPTG at 16-18°C overnight

• Purification approach:

  • For His-tagged constructs: Ni-NTA affinity chromatography

  • Follow with size exclusion chromatography to ensure homogeneity

  • Typical buffer composition: 50 mM Tris-HCl pH 7.5-8.0, 300 mM NaCl, 5-10% glycerol

• Quality control:

  • SDS-PAGE and Western blot analysis

  • Mass spectrometry verification

  • Functional assays if applicable

Since ribosomal proteins can form inclusion bodies when overexpressed, researchers might need to optimize solubility by using solubility-enhancing tags or refolding protocols.

What sequencing approaches are most appropriate for analyzing B. henselae rpsQ genetic diversity?

Based on the search results, several sequencing approaches have been demonstrated to be effective for analyzing genetic diversity in Bartonella species, which could be applied to rpsQ analysis:

• Sanger sequencing:

  • Traditional approach for single-gene analysis

  • Suitable for rpsQ gene sequencing from pure cultures or PCR products

  • Can detect single nucleotide polymorphisms (SNPs) with high accuracy

• Next-Generation Sequencing (NGS):

  • Amplicon-based NGS has been used successfully for Bartonella detection

  • Using Illumina MiSeq with 250 bp paired-end reads provides high-quality data for variant analysis

  • Allows detection of minority variants that might be missed by Sanger sequencing

• Multilocus Sequence Typing (MLST):

  • rpsQ could potentially be incorporated into MLST schemes

  • Current MLST approaches for B. henselae use nine genes and can distinguish seven genotypes

  • Adding rpsQ to existing schemes could potentially increase discriminatory power

• Multispacer Typing (MST):

  • Has been shown to be more discriminatory than MLST for B. henselae, identifying 39 genotypes among 126 isolates

  • This method focuses on intergenic spacers rather than genes

  • Could be complementary to rpsQ-based analysis

The choice of sequencing approach should depend on research objectives, available resources, and required resolution of genetic diversity.

How can rpsQ be utilized in developing diagnostic assays for B. henselae infections?

The development of diagnostic assays targeting B. henselae rpsQ could leverage several approaches:

• LAMP-based detection:

  • LAMP has demonstrated high sensitivity for Bartonella detection (125 fg/reaction), outperforming qPCR in clinical sample testing

  • For rpsQ-based LAMP assay development, researchers should:

    • Design specific primers targeting conserved regions of rpsQ

    • Optimize reaction conditions (temperature, time, reagent concentrations)

    • Validate against related species to ensure specificity

    • Test with clinical samples to determine sensitivity and specificity in real-world conditions

• PCR-based methods:

  • Various PCR approaches have been used for Bartonella detection, including broad-range PCR amplification of the 16S rRNA gene

  • For rpsQ-specific assays, researchers could develop:

    • Conventional PCR with species-specific primers

    • Real-time PCR with hybridization probes for increased sensitivity

    • Multiplex PCR incorporating rpsQ and other targets for increased specificity

• Sensitivity and specificity considerations:

  • In comparative studies of detection methods for Bartonella, LAMP demonstrated significantly higher sensitivity than qPCR when testing rhesus blood samples (22% vs. 8% positivity) and rhesus-feeder blood samples (20% vs. 5%)

  • Similar performance advantages might be achieved with rpsQ-targeted assays

What role might rpsQ play in B. henselae's adaptation to different hosts?

As a ribosomal protein, rpsQ is fundamentally involved in protein synthesis, which could potentially influence B. henselae's ability to adapt to different host environments:

• Host-specific adaptation:

  • B. henselae has a complex relationship between cat and human isolates. In some geographic regions, human isolates predominantly belong to 16S rRNA gene type I while cat isolates are mostly type II; in other regions, the pattern is reversed

  • Similar host-specific patterns might be detectable in rpsQ sequences or expression levels

  • Comparative analysis of rpsQ sequences from human vs. cat isolates could reveal selection pressures related to host adaptation

• Potential mechanisms:

  • Alterations in rpsQ might influence translation efficiency of specific mRNAs

  • Post-translational modifications of rpsQ could vary between host environments

  • Expression regulation of rpsQ might differ in response to host-specific stressors

• Research directions:

  • Comparative genomic analysis of rpsQ across isolates from different hosts

  • Experimental studies examining rpsQ expression under conditions mimicking different host environments

  • Investigation of potential post-translational modifications of rpsQ in different conditions

How does B. henselae rpsQ interact with other components of the bacterial ribosome?

Understanding the structural interactions of rpsQ within the B. henselae ribosome requires integrating general knowledge of bacterial ribosome structure with Bartonella-specific research:

• Predicted structural interactions:

  • The 30S ribosomal protein S17 typically interacts with the 16S rRNA and neighboring ribosomal proteins

  • In most bacteria, S17 is positioned at the interface between the head and platform of the 30S subunit

  • Key interaction partners likely include the 16S rRNA and ribosomal proteins S5, S9, and S20

• Functional implications:

  • These interactions are crucial for ribosome assembly and stability

  • Alterations in rpsQ structure could potentially affect translation efficiency or accuracy

  • Species-specific variations might reflect adaptations to different environmental conditions

• Research approaches:

  • Cryo-electron microscopy to determine B. henselae ribosome structure

  • Cross-linking studies to identify specific interaction partners

  • Mutagenesis studies to evaluate the impact of specific residues on ribosome function

What control strategies should be implemented when studying recombinant B. henselae rpsQ?

Robust experimental design for recombinant rpsQ studies should include multiple control strategies:

• Positive and negative controls for expression studies:

  • Positive control: Well-characterized recombinant protein expressed in the same system

  • Negative control: Host cells transformed with empty vector

  • Expression time course to determine optimal induction conditions

• Controls for functional studies:

  • Wild-type rpsQ protein (if available)

  • Structurally similar ribosomal proteins from related species

  • Site-directed mutants affecting key functional residues

• Controls for interaction studies:

  • No-bait controls in pull-down experiments

  • Competitive inhibition controls

  • Non-related proteins of similar size and charge properties

• Validation approaches:

  • Multiple detection methods (e.g., antibody-based and MS-based)

  • Replication across different experimental conditions

  • In vitro versus in vivo validation

How can researchers overcome challenges in analyzing rpsQ expression data from infected tissues?

Analysis of rpsQ expression in infected tissues presents several challenges that can be addressed with specialized approaches:

• Challenge: Low abundance of bacterial transcripts relative to host RNA

  • Solution: Enrichment strategies such as:

    • Selective capture of transcribed sequences (SCOTS)

    • Host RNA depletion methods

    • Bacterial-specific primer designs for RT-PCR

• Challenge: Distinguishing specific B. henselae strains

  • Solution: Strain-specific primer designs targeting SNPs in rpsQ

  • NGS approaches to identify minority variants

  • Digital PCR for absolute quantification of specific variants

• Challenge: Heterogeneous infection patterns in tissues

  • Solution: Laser capture microdissection to isolate specific infected regions

  • Single-cell RNA-seq to analyze bacteria-host interactions at the cellular level

  • Spatial transcriptomics to map expression patterns across tissue sections

• Challenge: Normalizing expression data

  • Solution: Use multiple reference genes validated for stability in infection conditions

  • Consider normalization to bacterial genome copy number rather than housekeeping genes

  • Apply advanced normalization algorithms designed for host-pathogen dual RNA-seq

What are the most effective approaches for analyzing potential post-translational modifications of B. henselae rpsQ?

Investigation of post-translational modifications (PTMs) on rpsQ requires specialized analytical techniques:

• Mass spectrometry-based approaches:

  • Bottom-up proteomics: Enzymatic digestion followed by LC-MS/MS

  • Top-down proteomics: Analysis of intact protein to preserve PTM combinations

  • Targeted approaches using multiple reaction monitoring (MRM) for specific modifications

• Enrichment strategies for specific PTMs:

  • Phosphorylation: Titanium dioxide or IMAC enrichment

  • Glycosylation: Lectin affinity chromatography

  • Ubiquitination/SUMOylation: Antibody-based enrichment

• Complementary approaches:

  • Western blotting with modification-specific antibodies

  • 2D gel electrophoresis to separate modified proteoforms

  • Chemical labeling strategies (e.g., SILAC, TMT) for quantitative comparison

• Bioinformatic analysis:

  • PTM site prediction using algorithms specific to bacterial proteins

  • Structural modeling to assess the impact of identified PTMs

  • Comparative analysis across different growth conditions or strains

How can contradictory results in B. henselae rpsQ research be reconciled?

When facing contradictory findings in rpsQ research, several analytical approaches can help reconcile discrepancies:

• Methodological differences analysis:

  • Systematically compare experimental methods used across studies

  • Evaluate differences in strains, growth conditions, and analytical techniques

  • Consider method-specific biases or limitations

• Strain and genetic variability assessment:

  • B. henselae shows significant genetic diversity, with MST identifying 39 genotypes among 126 studied isolates

  • Differences in rpsQ function or expression might reflect strain-specific variations

  • Geographic distribution of strains may explain regional differences in findings

• Integration of multiple data types:

  • Combine genomic, transcriptomic, and proteomic data to build a comprehensive picture

  • Consider environmental and host factors that might influence results

  • Apply systems biology approaches to model complex interactions

• Meta-analysis strategies:

  • Formal statistical meta-analysis of quantitative results

  • Development of standardized protocols to reduce methodological variation

  • Collaborative cross-laboratory validation studies

How does the study of rpsQ contribute to understanding the evolution of the Bartonella genus?

The evolutionary significance of rpsQ in Bartonella can be examined through several research lenses:

• Phylogenetic analysis:

  • rpsQ sequences can be incorporated into multi-gene phylogenies

  • Comparison with established phylogenetic markers (16S rRNA, gltA, rpoB, groEL) can reveal congruence or conflict in evolutionary signals

  • Analysis of selection pressure (dN/dS ratios) can identify regions under purifying or diversifying selection

• Horizontal gene transfer assessment:

  • MLST studies have suggested that lateral gene transfer occurs among B. henselae isolates

  • Analysis of rpsQ and flanking regions could reveal evidence of recombination events

  • Comparison with other Bartonella species might identify instances of interspecies gene transfer

• Host adaptation signatures:

  • Comparative analysis of rpsQ from isolates adapted to different hosts

  • Identification of convergent adaptations in lineages with similar host preferences

  • Assessment of correlation between rpsQ variants and host specificity

• Integration with current understanding:

  • Current typing methods have revealed that B. henselae is significantly more genetically diverse than B. quintana, which is mostly clonal

  • rpsQ analysis could help explain the mechanisms driving this differential diversity

What are the most promising future research directions involving B. henselae rpsQ?

Based on current knowledge and methodological capabilities, several promising research directions emerge:

• Structural biology approaches:

  • Cryo-EM studies of the B. henselae ribosome to determine the precise structural role of rpsQ

  • X-ray crystallography of isolated rpsQ to identify potential binding sites

  • Structural comparison across Bartonella species to identify conserved functional elements

• Systems biology integration:

  • Network analysis incorporating rpsQ interactions with other cellular components

  • Multi-omics approaches to place rpsQ in its broader cellular context

  • Mathematical modeling of ribosome assembly and function with rpsQ variants

• Translational research:

  • Development of rpsQ-based diagnostic tools leveraging the sensitivity of methods like LAMP

  • Exploration of rpsQ as a potential drug target

  • Vaccine development strategies incorporating rpsQ epitopes

• Evolutionary medicine:

  • Analysis of rpsQ in the context of B. henselae adaptation to human hosts

  • Investigation of rpsQ variants in treatment-resistant isolates

  • Study of rpsQ expression under antibiotic pressure