Recombinant Rickettsia typhi Aspartate--tRNA ligase (aspS), partial

What is the genomic context of the aspS gene in Rickettsia typhi?

The R. typhi genome is highly syntenic across different isolates, with relatively few single nucleotide polymorphisms (SNPs) and insertion-deletion (INDEL) sites . While specific information about the aspS locus is limited in the provided data, the gene likely exists in a conserved genomic region given the minimal genetic diversity observed across R. typhi isolates from different geographical regions. Comparisons of complete genome sequences from North Carolina (Wilmington), Myanmar (B9991PP), and Thailand (TH1527) identified only 26 SNP and 7 INDEL sites, demonstrating remarkable genetic stability . This conservation suggests essential genes like aspS are under strong purifying selection.

How does R. typhi Aspartate--tRNA ligase compare structurally to orthologs in other bacterial species?

Aspartate--tRNA ligase belongs to the class II aminoacyl-tRNA synthetase family. Though specific structural information for R. typhi aspS is not provided in the search results, image mentions a crystal structure of Methionyl-tRNA Synthetase from R. typhi, suggesting structural studies have been performed on related synthetases. Researchers should note that aminoacyl-tRNA synthetases in bacteria often display conserved catalytic domains while exhibiting species-specific features in non-catalytic regions that may contribute to specialized functions beyond translation.

What methods are available for cloning and expressing the R. typhi aspS gene?

Methodological approach: Similar to the approach used for R. typhi ankyrin repeat protein (RARP-1), researchers can amplify the aspS gene using PCR from genomic DNA isolated from cultured R. typhi . The gene can then be cloned into expression vectors such as pET or pBAD systems. Due to the obligate intracellular nature of Rickettsia, heterologous expression in E. coli is typically employed, as seen with other rickettsial proteins . When designing expression constructs, researchers should consider:

• Codon optimization for the host expression system

• Inclusion of purification tags (His, GST, etc.)

• Testing multiple expression conditions (temperature, induction time)

• Employing specialized E. coli strains (e.g., Rosetta for rare codon usage)

How can researchers assess the enzymatic activity of recombinant R. typhi Aspartate--tRNA ligase?

Methodological approach: Aminoacyl-tRNA synthetase activity can be measured through several techniques:

• ATP-pyrophosphate exchange assay: Measuring the incorporation of radioactive pyrophosphate into ATP during the amino acid activation step

• tRNA aminoacylation assay: Monitoring the charging of tRNA^Asp with radiolabeled aspartate

• Coupled enzyme assays: Using pyrophosphatase and monitoring inorganic phosphate release
  Similar to studies on R. typhi phospholipases, researchers may need to investigate whether the enzyme requires host cofactors for optimal activity . R. typhi Pat1 and Pat2 phospholipases both required host cofactors for enzymatic activity, suggesting this may be a common feature of rickettsial enzymes that interface with host components .

What role might aspS play in R. typhi pathogenesis and host-pathogen interactions?

While direct evidence for aspS involvement in pathogenesis is not provided in the search results, several rickettsial proteins are secreted into host cells to modulate cellular functions. For example, RARP-1 and phospholipases (Pat1/Pat2) are secreted into the host cytoplasm during infection . AspS could potentially:

• Function in its canonical role in protein synthesis within the pathogen

• Be secreted into host cells to perform moonlighting functions

• Serve as an immunogenic antigen recognized by the host
  To investigate potential non-canonical roles, researchers should:

• Perform localization studies using immunofluorescence assays (IFA) similar to those used for Pat1/Pat2

• Use immunoblotting to detect aspS in host cytoplasmic fractions during infection

• Employ antibody-blocking experiments to assess the enzyme's role during invasion

How can researchers differentiate between host and pathogen aminoacyl-tRNA synthetases in experimental settings?

Methodological approach: This distinction is crucial for studying pathogen-specific processes. Researchers can:

• Design specific antibodies against unique epitopes of R. typhi aspS

• Develop specific PCR primers for discriminating between host and pathogen transcripts

• Use mass spectrometry-based approaches with peptide signatures unique to the rickettsial enzyme

• Employ genetic approaches using tagged versions of the rickettsial protein
  This approach follows similar principles used in differentiating R. typhi from R. prowazekii, where specific genetic markers were developed to distinguish between closely related species .

What are the challenges in purifying active recombinant R. typhi Aspartate--tRNA ligase?

Common challenges include:

• Protein solubility issues: Rickettsial proteins may form inclusion bodies when expressed in E. coli

• Protein stability: The enzyme may require specific buffer conditions or additives

• Co-purification of contaminating E. coli proteins

• Requirement for host cofactors for proper folding or activity
  Methodological solutions:

• Test multiple expression conditions (temperature, induction time)

• Use solubility-enhancing fusion tags (MBP, SUMO)

• Optimize lysis and purification buffers with stabilizing agents

• Consider on-column refolding protocols for proteins recovered from inclusion bodies
  Similar approaches have been successful for other rickettsial proteins, such as the Pat1 and Pat2 phospholipases, which were expressed as recombinant proteins and demonstrated enzymatic activity in vitro .

How can researchers investigate potential secretion of aspS into host cells during infection?

The search results describe several R. typhi proteins secreted into host cells, including Pat1, Pat2, and RARP-1 . If aspS is hypothesized to be secreted, researchers could:

• Perform immunofluorescence assays using anti-aspS antibodies during different stages of infection

• Use cell fractionation and immunoblotting to detect aspS in host cytoplasmic fractions

• Employ TEM-immunogold labeling to visualize aspS localization at the ultrastructural level
  It's worth noting that R. typhi proteins like Pat1 and Pat2 lack conventional Sec-dependent signal sequences yet are still secreted into host cells . This suggests unconventional secretion mechanisms may be at play for multiple rickettsial proteins. The TolC secretion system (type 1 secretion system) has been implicated in the secretion of RARP-1 in R. typhi and could potentially be involved in aspS secretion as well .

What approaches can be used to identify potential inhibitors of R. typhi Aspartate--tRNA ligase for antimicrobial development?

Methodological approach:

• High-throughput screening assays:

  • ATP-pyrophosphate exchange assays in 96-well format

  • Fluorescence-based aminoacylation assays using labeled tRNAs

  • Fragment-based screening approaches

• Structure-based drug design:

  • If crystal structures are available, perform in silico docking studies

  • Focus on the active site and species-specific pockets

  • Design compounds that exploit differences between bacterial and human enzymes

• Validation and secondary assays:

  • Confirm hits with dose-response curves

  • Test specificity against human cytoplasmic and mitochondrial AspRS

  • Evaluate cellular activity using cell culture infection models

How do evolutionary patterns of aspS compare with other genes in Rickettsia typhi?

Evolutionary analysis of R. typhi genes reveals different patterns. For instance, phospholipase genes pat1 and pat2 show divergent evolutionary histories, with pat2 being deleted in many non-Typhus Group rickettsiae . While specific data on aspS evolution is not provided, essential genes like aminoacyl-tRNA synthetases typically show high conservation. Researchers investigating aspS evolution should:

• Perform phylogenetic analysis across rickettsial species

• Calculate selection pressures (dN/dS ratios) to identify conserved functional domains

• Look for evidence of horizontal gene transfer or recombination events
  Similar to pat1, which shows evidence of recombination with plasmid-encoded homologs, researchers should examine whether aspS has undergone recombination events during rickettsial evolution .

What techniques can be used to study potential interactions between R. typhi Aspartate--tRNA ligase and host factors?

Methodological approach:

• Affinity purification coupled with mass spectrometry:

  • Express tagged aspS in mammalian cells or during infection

  • Purify the protein complex and identify interacting partners

• Yeast two-hybrid or bacterial two-hybrid screening:

  • Use aspS as bait to screen against host protein libraries

  • Validate interactions using co-immunoprecipitation

• Proximity labeling approaches:

  • Fuse aspS to BioID or APEX2 for proximity-dependent labeling

  • Identify proximal proteins during infection

• Biophysical techniques:

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Microscale thermophoresis for quantifying interactions
    This approaches align with studies of other R. typhi proteins that interact with host components, such as the phospholipases that require host cofactors for activity .

How might genomic and phenotypic variation in R. typhi isolates affect aspS function across strains?

• Examining whether any SNPs or INDELs affect the aspS coding sequence or regulatory regions

• Comparing aspS expression levels across different R. typhi isolates

• Investigating whether aspS enzyme kinetics vary between strains from different geographical regions
  The study of genetic typing of R. typhi isolates provides a foundation for understanding strain-specific variations that might impact aspS function .

What potential exists for developing aspS-based diagnostic tests for R. typhi infection?

R. typhi diagnosis currently relies on serological methods or PCR. Given the genetic stability observed across R. typhi isolates , aspS could serve as a target for:

• PCR-based detection methods:

  • Design specific primers targeting conserved regions of aspS

  • Develop quantitative PCR assays for pathogen load determination

• Serological detection:

  • Evaluate whether aspS generates antibody responses during infection

  • Develop recombinant aspS-based ELISA assays

• CRISPR-based diagnostics:

  • Design Cas13-based detection systems targeting aspS transcripts

  • Develop paper-based diagnostic tests for field use
    Methodological considerations should include analysis of aspS conservation across strains and specificity relative to other Rickettsia species, particularly R. prowazekii, its closest relative .