Recombinant Neisseria meningitidis serogroup C Elongation factor Ts (tsf)

What is Neisseria meningitidis serogroup C and how does it differ from other serogroups?

Neisseria meningitidis is a bacterium that causes meningococcal disease, with serogroup C being one of the five major disease-causing serogroups (along with A, B, Y, and W). These five serogroups cause almost all invasive meningococcal disease globally . Serogroup classification is primarily based on the chemical composition of the bacterial capsular polysaccharide. Unlike serogroup A (which consists of repeating units of N-acetyl-mannosamine-1-phosphate), serogroup C's capsule is composed of sialic acid derivatives .

In the United States, serogroups C, W, and Y account for more than half of reported meningococcal disease cases . The genome of N. meningitidis serogroup C (strain FAM18) has been fully sequenced and is approximately 2.0-2.2 megabases in size, containing about 2,000 genes . The capsule is essential for bacterial survival in the bloodstream as it provides resistance to antibody/complement-mediated killing and inhibits phagocytosis .

What is the molecular function of Elongation factor Ts (tsf) in bacterial protein synthesis?

Elongation factor Ts (EF-Ts), encoded by the tsf gene, is a guanine nucleotide exchange factor critical for bacterial protein synthesis. While the provided search results don't specifically detail EF-Ts function in N. meningitidis, bacterial translation is highly conserved across species. EF-Ts catalyzes the regeneration of active EF-Tu by facilitating the exchange of GDP for GTP, allowing EF-Tu to continue delivering aminoacyl-tRNAs to the ribosome during translation elongation.

By analogy with other essential translation factors in N. meningitidis, such as Translation elongation factor P (EF-P) , EF-Ts likely plays a crucial role in protein synthesis and bacterial viability. The efp gene encoding EF-P has been demonstrated to be essential for N. meningitidis survival, as attempts to disrupt this gene were unsuccessful without a complementary functional copy .

How do researchers isolate and characterize functional tsf from N. meningitidis serogroup C?

To isolate and characterize functional tsf from N. meningitidis serogroup C, researchers can employ methodologies similar to those used for other meningococcal proteins. The approach typically involves:

• Gene amplification and cloning: The tsf gene can be PCR-amplified from N. meningitidis serogroup C genomic DNA using specific primers. Similar to approaches used for other meningococcal proteins, the gene can then be cloned into a suitable expression vector.

• Heterologous expression: Expression in E. coli is commonly used for meningococcal proteins, as demonstrated with transferrin binding proteins (TbpA and TbpB) . Optimization of expression conditions (temperature, induction time, media) is crucial to obtain soluble protein.

• Protein purification: Affinity chromatography using tags (such as His-tag) followed by size exclusion chromatography can be employed to purify the recombinant protein. For TbpA and TbpB, affinity chromatography using human transferrin enabled purification of functional proteins .

• Functional characterization: Activity assays specific to EF-Ts function, such as nucleotide exchange assays with EF-Tu, can verify the functionality of the purified protein. Structural studies using X-ray crystallography or cryo-electron microscopy may provide insights into the protein's three-dimensional structure.

What are the optimal PCR conditions for amplifying the tsf gene from N. meningitidis serogroup C?

For optimal PCR amplification of the tsf gene from N. meningitidis serogroup C, researchers should consider approaches similar to those developed for the detection of other meningococcal genes. Based on the real-time PCR methodology developed for sodC , researchers might consider:

• Primer design: Design primers specific to conserved regions of the tsf gene in N. meningitidis serogroup C. Analyzing sequence data from multiple strains can help identify highly conserved regions suitable for primer binding.

• PCR conditions: For the sodC-based TaqMan real-time PCR assay, researchers achieved high sensitivity and specificity with optimized cycling conditions . Similar optimization would be needed for tsf amplification.

• Reaction components: The reaction mixture should contain appropriate concentrations of primers, DNA polymerase, dNTPs, and magnesium chloride. For the sodC assay, researchers achieved a lower limit of detection of 73 genomes per reaction .

• Validation: Similar to the sodC assay validation, which tested 626 N. meningitidis isolates and 244 non-N. meningitidis isolates , extensive validation with diverse strain collections would be necessary to ensure the reliability of tsf amplification.

When adapting these methods for conventional PCR rather than real-time PCR, appropriate adjustments to cycling conditions and detection methods would be required.

How does the genetic sequence of tsf vary among different strains of N. meningitidis serogroup C?

To thoroughly characterize tsf variation, researchers could perform comparative genomic analyses across multiple serogroup C isolates, potentially identifying conserved regions suitable for universal primers and variable regions that might affect protein function or antigenicity.

What genetic elements regulate the expression of tsf in N. meningitidis under different environmental conditions?

While the search results don't specifically address the regulation of tsf expression in N. meningitidis, the regulation of essential genes in bacteria often responds to environmental changes. For tsf regulation, researchers might investigate:

• Promoter architecture: Identifying the promoter elements upstream of the tsf gene would provide insights into its basal expression level and potential regulatory mechanisms.

• Transcriptional regulation: Essential genes often have complex regulatory mechanisms ensuring appropriate expression levels. Potential regulators might include global transcription factors responding to stress, nutrient availability, or growth phase.

• Post-transcriptional regulation: RNA-based regulation, including small RNAs or riboswitches, might modulate tsf mRNA stability or translation efficiency in response to environmental conditions.

• Experimental approaches: Techniques such as reporter gene fusions, qRT-PCR under various conditions, and chromatin immunoprecipitation could help elucidate the regulatory mechanisms controlling tsf expression.

Understanding these regulatory mechanisms could provide insights into how N. meningitidis adapts its translation machinery during infection and in response to environmental stresses, potentially revealing new targets for therapeutic intervention.

What is the three-dimensional structure of N. meningitidis Elongation factor Ts, and how does it compare to homologs from other bacteria?

• Domain organization: Bacterial EF-Ts typically consists of an N-terminal domain, a core domain containing the EF-Tu binding site, and a C-terminal domain. The relative orientations of these domains can vary between species.

• Structural determination methods: To determine the structure of N. meningitidis EF-Ts, researchers could employ X-ray crystallography or cryo-electron microscopy. Expression and purification protocols similar to those used for other N. meningitidis proteins would be the first step .

• Comparative structural analysis: Once determined, the structure could be compared to EF-Ts from other pathogens and model organisms to identify conserved functional regions and species-specific features that might be targeted for antimicrobial development.

• Structure-function relationships: Correlating structural features with functional data would help identify critical residues involved in nucleotide exchange activity and interactions with EF-Tu.

This structural information would be valuable for understanding the molecular mechanism of translation in N. meningitidis and could guide structure-based drug design efforts targeting this essential protein.

What biochemical assays can measure the nucleotide exchange activity of recombinant tsf?

To assess the nucleotide exchange activity of recombinant N. meningitidis EF-Ts, researchers can employ several biochemical assays:

• Radiometric nucleotide exchange assay: This classic approach measures the exchange of radiolabeled GDP/GTP on EF-Tu catalyzed by EF-Ts. The reaction typically involves preloading EF-Tu with [³H]GDP, then measuring the release of radioactivity in the presence of EF-Ts and excess unlabeled GTP.

• Fluorescent nucleotide analogs: Using fluorescent GDP/GTP analogs (such as mant-GDP) allows real-time monitoring of nucleotide exchange through changes in fluorescence intensity or anisotropy when the nucleotide binds to or dissociates from EF-Tu.

• Biolayer interferometry or surface plasmon resonance: These techniques can measure the kinetics of EF-Ts binding to EF-Tu·GDP and the subsequent dissociation of the EF-Ts·EF-Tu complex upon GTP addition.

• Coupled enzymatic assays: The GDP release can be coupled to enzymatic reactions that produce a measurable signal, allowing continuous monitoring of exchange activity.

• In vitro translation assays: While less direct, these assays can assess the functional impact of EF-Ts on translation efficiency in reconstituted systems.

For all these assays, careful optimization of reaction conditions (pH, temperature, salt concentration) is necessary to accurately measure kinetic parameters and compare activities between wild-type and mutant proteins or between species.

How do specific mutations in the tsf gene affect protein function and N. meningitidis viability?

• Conditional mutagenesis: Similar to the approach used for EF-P , researchers could generate N. meningitidis strains with an inducible copy of wild-type tsf while introducing mutations in the chromosomal copy. Growth in the presence and absence of inducer would reveal whether specific mutations affect viability.

• Site-directed mutagenesis: Based on structural and sequence conservation data, researchers could introduce specific mutations in recombinant tsf and assess their impact on nucleotide exchange activity using the biochemical assays described above.

• Complementation studies: Testing whether mutant versions of tsf can complement growth defects in conditional tsf mutants would provide insights into which protein regions are critical for function in vivo.

• Structural mapping: Correlating functional data with structural information would help identify critical functional domains and potential sites for targeted drug development.

The study of EF-P in N. meningitidis demonstrated that when the efp gene contained a premature stop codon or was deleted, growth was severely impaired or abolished without a complementary functional copy , highlighting the potential essentiality of translation factors like tsf.

How can recombinant tsf be used to develop novel diagnostic tools for N. meningitidis detection?

Recombinant tsf could potentially be utilized for diagnostic applications through several approaches:

• PCR-based detection: If the tsf gene contains regions unique to N. meningitidis, it could serve as a molecular target for PCR-based detection. The success of sodC-based PCR, which achieved 99.7% sensitivity and 100% specificity for N. meningitidis detection , provides a model for developing and validating such assays.

• Antibody development: Recombinant tsf could be used to generate specific antibodies for immunological detection methods. These antibodies could be employed in various formats, including ELISA, lateral flow assays, or immunofluorescence microscopy.

• Aptamer selection: Recombinant tsf could serve as a target for selecting DNA or RNA aptamers with high affinity and specificity, which could then be incorporated into biosensor platforms.

• Comparative advantage assessment: Any new tsf-based diagnostic would need to demonstrate advantages over existing methods like sodC-PCR, which already shows excellent performance in detecting both capsulated and noncapsulated N. meningitidis isolates .

The potential advantage of tsf-based diagnostics might lie in the ability to differentiate between pathogenic and non-pathogenic Neisseria species or to identify specific serogroups if sufficient sequence variation exists in the tsf gene across these categories.

What high-throughput screening methods can identify inhibitors of N. meningitidis tsf?

To identify potential inhibitors of N. meningitidis EF-Ts, researchers could employ several high-throughput screening approaches:

• Biochemical activity assays: Adapting the nucleotide exchange assays described earlier to microplate format would allow screening of compound libraries for inhibitors that reduce EF-Ts activity. Fluorescence-based assays would be particularly amenable to high-throughput formats.

• Thermal shift assays: Also known as differential scanning fluorimetry, this technique can identify compounds that bind to and stabilize (or destabilize) EF-Ts, causing shifts in the protein's melting temperature.

• Fragment-based screening: NMR or X-ray crystallography can be used to identify small molecule fragments that bind to EF-Ts, which can then be expanded or linked to develop more potent inhibitors.

• Virtual screening: If the three-dimensional structure of N. meningitidis EF-Ts is available, computational docking of virtual compound libraries can identify potential binding molecules for subsequent experimental validation.

• Whole-cell screening: Compounds can be screened for selective growth inhibition of N. meningitidis, with target validation performed subsequently to confirm EF-Ts as the target.

Given the essential nature of translation for bacterial viability, as demonstrated for EF-P in N. meningitidis , inhibitors of EF-Ts could potentially serve as novel antimicrobials against this pathogen.

How does the essentiality of tsf compare with other potential drug targets in N. meningitidis?

While the essentiality of tsf in N. meningitidis is not directly addressed in the provided search results, we can draw parallels with other essential genes and processes:

• Translation factors: The essentiality of EF-P in N. meningitidis has been demonstrated through unsuccessful attempts to disrupt the efp gene without complementation . As another translation factor, tsf might share this essential nature.

• Comparative genomics: The core meningococcal genome represents about 70% of the total genome and encodes essential metabolic functions . Determining where tsf ranks among essential genes would require comprehensive gene essentiality studies.

• Target validation approaches: Methods to assess essentiality include:

  • Conditional gene expression systems, as used for EF-P

  • CRISPR interference to gradually reduce gene expression

  • Antisense RNA approaches to modulate expression levels

• Vulnerability assessment: Beyond binary essentiality, the concept of "vulnerability" considers how much a protein's activity can be reduced before growth is affected. Quantitative studies of tsf expression levels and cellular requirements would provide this information.

• Drug target criteria: Ideal drug targets combine essentiality with other favorable properties:

  • No close human homolog or significant structural differences from human homologs

  • Accessibility to inhibitors (though this is less critical for cytoplasmic targets)

  • Low propensity for resistance development

Comparing tsf with other essential proteins across these criteria would help prioritize drug development efforts against N. meningitidis.

What is the immunogenicity profile of recombinant N. meningitidis tsf in animal models?

These studies would provide crucial data on whether tsf has potential as a vaccine antigen despite its presumed cytoplasmic location.

How do differential immunization strategies affect the quality of immune responses to recombinant tsf?

Different immunization strategies can significantly impact the quality and protective efficacy of immune responses to bacterial antigens. Based on studies of other meningococcal antigens, researchers investigating tsf would likely examine:

• Adjuvant selection: Different adjuvants can skew immune responses toward Th1, Th2, or mixed profiles. The choice of adjuvant could be critical for inducing protective immunity against N. meningitidis.

• Route of administration: Intramuscular, subcutaneous, intranasal, or other routes may generate qualitatively different immune responses, potentially affecting mucosal immunity—relevant for a pathogen that colonizes the nasopharynx.

• Prime-boost strategies: Heterologous prime-boost approaches (using different formulations or delivery systems for initial and booster immunizations) might enhance both antibody and T-cell responses.

• Antigen presentation formats: Presenting tsf in different contexts could affect immunogenicity:

  • Soluble recombinant protein

  • Protein conjugated to carrier molecules

  • DNA vaccines encoding tsf

  • Viral vector vaccines expressing tsf

• Combination approaches: Co-administration with other meningococcal antigens might provide synergistic effects, as could be explored by combining tsf with established vaccine antigens.

For all these strategies, comprehensive immune profiling and protection studies would be essential to determine which approach elicits the most effective and durable immune responses against N. meningitidis.

What methods can determine if anti-tsf antibodies confer protection across different N. meningitidis strains?

To assess the cross-protective potential of anti-tsf antibodies against diverse N. meningitidis strains, researchers could employ methodologies similar to those used for other meningococcal antigens:

• Serum bactericidal assays (SBA): Test the ability of anti-tsf antibodies to kill different N. meningitidis strains in the presence of complement. The study of TbpA and TbpB showed varying bactericidal activity against different strains .

• Cross-strain protection studies: Challenge immunized animals with different N. meningitidis strains to assess protection breadth. TbpA immunization protected against both homologous and heterologous strains, including a serogroup C isolate .

• Sequence and structure analysis: Compare tsf sequences across strains to identify conserved regions that might elicit cross-protective antibodies. Higher conservation would suggest broader protection potential.

• Epitope mapping: Identify specific regions (epitopes) of tsf recognized by protective antibodies. Determining whether these epitopes are conserved across strains would predict cross-protection.

• Opsonophagocytic assays: Evaluate whether anti-tsf antibodies enhance phagocytosis of various N. meningitidis strains by neutrophils or macrophages.

• Passive immunization studies: Transfer anti-tsf sera or purified antibodies to naïve animals and challenge with different strains to directly assess cross-protection.

The TbpA study demonstrated that protection mechanisms may not always correlate with serum bactericidal activity , highlighting the importance of using multiple approaches to evaluate potential cross-protection.