Recombinant Streptococcus pneumoniae Translation initiation factor IF-2 (infB), partial

What is Streptococcus pneumoniae Translation initiation factor IF-2 (infB)?

Translation initiation factor IF-2 (infB) is a critical protein encoded by the infB gene in Streptococcus pneumoniae that facilitates the initiation of protein synthesis. The protein exists in two distinct forms: IF2 alpha (97,300 daltons) and IF2 beta (79,700 daltons), which differ at their N-terminus but serve complementary roles in translation initiation . In the strain CGSP14, the full-length protein spans amino acids 1-958 and functions as a key component in the bacterial translational machinery, helping to position the initiator tRNA on the ribosome during the formation of the initiation complex .

How does S. pneumoniae infB compare to translation initiation factors in other bacterial species?

S. pneumoniae infB shares structural homology with translation initiation factors across bacterial species but displays unique characteristics particularly relevant to clinical research. Unlike some bacterial species with a single IF2 variant, S. pneumoniae produces two distinct forms (alpha and beta) that are independently translated from different translation initiation sites on the same gene, rather than resulting from post-translational modification . This dual initiation mechanism represents an important regulatory feature that may contribute to the organism's adaptability under different environmental conditions and stress responses.

What is the biological significance of the two forms of infB protein in S. pneumoniae?

The two forms of infB protein (IF2 alpha and IF2 beta) in S. pneumoniae result from translation initiation at two distinct sites within the infB gene. Research involving Edman degradation has confirmed that the N-terminal amino acid sequences of these two forms are completely different but match perfectly with the DNA sequences at the beginning of the infB open reading frame and an in-phase region 471 bp downstream . This dual expression system likely represents a sophisticated regulatory mechanism that allows the bacterium to fine-tune translation under various environmental conditions. Deletion studies of the 5'-non-translated region, including the Shine/Dalgarno ribosomal binding site, demonstrate that IF2 beta expression persists even when IF2 alpha expression is abolished, confirming independent translation initiation rather than proteolytic processing .

What expression systems are most effective for producing recombinant S. pneumoniae infB protein?

Recombinant S. pneumoniae infB protein can be successfully expressed in multiple heterologous systems including E. coli, yeast, baculovirus, and mammalian cell lines . Each system offers distinct advantages: E. coli provides high yield and cost-effectiveness, making it suitable for structural studies; yeast systems offer appropriate eukaryotic post-translational modifications; baculovirus systems excel for larger proteins requiring complex folding; and mammalian expression systems provide the most authentic post-translational modifications when studying host-pathogen interactions. For basic biochemical characterization, E. coli-based expression is typically preferred due to its efficiency and established purification protocols, while functional studies examining host interactions may benefit from mammalian expression systems.

What are the methodological challenges in purifying full-length recombinant infB protein?

Purification of full-length recombinant infB protein presents several methodological challenges that researchers must address through optimized protocols:

• Protein solubility issues may arise due to the large size (958 amino acids) of the full-length protein

• Maintaining the native conformation during purification requires careful buffer optimization

• Separating the two forms (IF2 alpha and IF2 beta) necessitates high-resolution chromatographic techniques

• Preventing proteolytic degradation during expression and purification requires protease inhibitors

An effective purification strategy typically employs affinity chromatography utilizing histidine or GST tags, followed by ion-exchange chromatography and size-exclusion chromatography for final polishing. Researchers should validate purified protein functionality through GTP binding assays or translation initiation activity assessments to ensure that the recombinant protein retains its biological activity.

How can researchers effectively distinguish between the alpha and beta forms of infB in experimental settings?

To effectively distinguish between IF2 alpha (97,300 daltons) and IF2 beta (79,700 daltons) forms in experimental settings, researchers can employ several techniques:

• SDS-PAGE analysis: The molecular weight difference (~17,600 daltons) allows separation on 8-10% polyacrylamide gels

• N-terminal sequencing: Edman degradation reveals distinct N-terminal sequences for each form

• Immunoblotting: Form-specific antibodies raised against unique N-terminal peptides provide selective detection

• Mass spectrometry: Provides precise mass determination and peptide mapping

• Recombinant constructs: Creating fusion proteins with reporter tags (as demonstrated with β-galactosidase) allows in vivo tracking of each form's expression and distribution

When designing experiments to study form-specific functions, researchers should consider using deletion constructs that selectively express only one form, as demonstrated by the deletion of the 5'-non-translated region which eliminates IF2 alpha expression while maintaining IF2 beta production .

What methodologies are available for studying the functional role of infB in S. pneumoniae protein synthesis?

Several methodologies can be employed to study the functional role of infB in S. pneumoniae protein synthesis:

• In vitro translation assays: Researchers can use purified components to reconstitute the translation initiation complex and measure the activity of recombinant infB protein through dipeptide synthesis assays

• Ribosome binding assays: Using fluorescence anisotropy or surface plasmon resonance to quantify the interaction between infB and ribosomal components

• GTP hydrolysis assays: Measuring the GTPase activity of infB as a proxy for functional activity

• Mutational analysis: Creating point mutations or domain deletions to identify critical regions for function

• Comparative analysis: Studying the differential activities of IF2 alpha and IF2 beta forms under various physiological conditions

An integrated approach combining these methods provides comprehensive insight into infB's role in initiating protein synthesis under different environmental conditions and stress responses that S. pneumoniae encounters during infection.

What is the relationship between infB function and antibiotic resistance in S. pneumoniae?

Although the search results don't directly address the relationship between infB and antibiotic resistance, we can infer connections based on fundamental bacterial physiology:

• Translation machinery components, including initiation factors, are targets for several classes of antibiotics

• Mutations or modifications in translation factors can contribute to antibiotic resistance mechanisms

• The efficient protein synthesis facilitated by infB enables expression of resistance genes under selective pressure

• S. pneumoniae's capacity for DNA uptake and homologous recombination, which enables antibiotic resistance spread, may indirectly involve translation factors through the expression of competence genes

Research investigating potential links between infB variants and resistance profiles could reveal whether specific forms of the protein correlate with enhanced survival under antibiotic pressure, potentially informing new therapeutic approaches targeting translation initiation.

How does recombination affect genetic variability in the infB gene across S. pneumoniae lineages?

Recombination plays a significant role in generating genetic variability in S. pneumoniae, including potential diversity in the infB gene. S. pneumoniae can import DNA from other strains or even species through transformation and homologous recombination, which contributes to its genetic plasticity . This recombination capability has allowed the pneumococcus to evade clinical interventions such as antibiotics and pneumococcal conjugate vaccines . Studies in Malawi have demonstrated that recombination rates vary among capsule types, with both the amount of variation introduced by recombination relative to mutation (the relative rate) and the frequency of individual recombination events per isolate varying significantly across serotypes .

What methodologies can researchers use to detect recombination events in the infB gene region?

Researchers can employ several methodologies to detect recombination events in the infB gene region:

• Comparative genomic analysis: Analyzing sequence data from multiple isolates to identify regions with atypical SNP density

• Software tools like Gubbins: These tools identify regions with an atypically high density of SNPs using a sliding window approach, which can reveal potential recombination events

• Phylogenetic analysis: Constructing gene trees and comparing them with species trees to identify incongruences indicative of recombination

• Population genetic approaches: Calculating linkage disequilibrium and other population genetic parameters that can reveal historical recombination events

• Experimental approaches: Using marked strains to monitor DNA transfer in laboratory settings

The choice of methodology depends on whether researchers are examining historical recombination events in natural populations or conducting controlled laboratory experiments to understand mechanisms and rates of recombination.

How does capsule size influence recombination rates in S. pneumoniae, and what implications might this have for infB evolution?

Research has demonstrated a significant association between capsule size and recombination rates in S. pneumoniae. Larger capsules show positive association with recombination, contrary to the initial hypothesis that they might physically inhibit DNA uptake . This relationship persists even after correcting for collinearity with other capsular factors using multivariate analysis .

The implications for infB evolution are significant:

• Serotypes with larger capsules may experience higher rates of infB gene variation due to increased recombination frequency

• This could lead to serotype-specific variants of infB with potentially altered functional properties

• The differential recombination rates across serotypes may create distinct evolutionary trajectories for infB in different lineages

• Higher recombination rates could accelerate the spread of adaptive mutations in infB under selective pressure

The data from Malawi shows that both the relative rate of recombination (μr/m) and the frequency of recombination events vary significantly across sequence clusters and serotypes, as demonstrated in the following table:

These findings suggest that infB evolution may proceed at different rates across pneumococcal lineages, potentially contributing to functional divergence of this important translation factor .

What role might recombinant infB protein play in pneumococcal vaccine development?

Recombinant S. pneumoniae infB protein has potential applications in pneumococcal vaccine development strategies:

• As a protein antigen: The conserved nature of translation factors across strains makes infB a potential universal antigen target that could overcome the limitations of serotype-specific polysaccharide vaccines

• As a carrier protein: Conjugation of capsular polysaccharides to protein carriers enhances immunogenicity; infB could potentially serve as a pneumococcal-derived carrier

• For epitope mapping: Recombinant infB fragments can be used to identify immunodominant epitopes for rational vaccine design

• In reverse vaccinology approaches: Computational analysis of infB sequence may reveal surface-exposed regions that could serve as vaccine targets

While current pneumococcal vaccines primarily target capsular polysaccharides, the limitations of serotype replacement and the varying recombination rates across serotypes highlight the need for protein-based approaches that could provide broader protection . The recombinant infB protein available commercially is already noted as being useful for vaccine development .

What experimental controls are essential when working with recombinant S. pneumoniae infB protein in functional assays?

When conducting functional assays with recombinant S. pneumoniae infB protein, researchers should implement the following essential experimental controls:

• Negative controls:

  • Buffer-only controls to establish baseline measurements

  • Heat-inactivated protein to confirm activity loss upon denaturation

  • Mutant variants lacking GTP-binding capacity to confirm specificity

• Positive controls:

  • Commercially validated infB protein with known activity

  • E. coli IF2 as a functional homolog for comparative analysis

  • Native S. pneumoniae lysate to compare recombinant protein activity to natural levels

• Specificity controls:

  • Testing both alpha and beta forms separately to distinguish form-specific functions

  • Including other translation factors (IF1, IF3) to assess potential synergistic effects

  • Competition assays with specific antibodies to confirm activity attribution

• Technical validation:

  • Multiple protein concentrations to establish dose-dependency

  • Time-course experiments to determine kinetic parameters

  • Replicates across different protein preparations to ensure reproducibility

These controls help ensure that observed effects are specifically attributable to infB activity rather than experimental artifacts or contaminating factors from the expression system.

How can researchers address the challenge of serotype-specific differences when studying infB across S. pneumoniae strains?

Addressing serotype-specific differences when studying infB across S. pneumoniae strains requires a comprehensive approach:

• Comparative genomic analysis:

  • Sequence infB genes from multiple serotypes to identify conserved and variable regions

  • Map variations to functional domains to predict impact on protein function

  • Correlate sequence variations with recombination rates across serotypes

• Experimental strategies:

  • Express and purify infB from multiple serotypes to compare biochemical properties

  • Conduct domain-swapping experiments to identify functional differences

  • Use site-directed mutagenesis to assess the impact of serotype-specific variations

• Epidemiological considerations:

  • Include strains from diverse geographical regions, as recombination patterns may vary

  • Consider the epidemiological relevance of serotypes, particularly those prevalent in resource-poor settings

  • Track changes in infB sequences following vaccine introduction to assess selection pressure

• Functional assessment:

  • Compare translation initiation efficiency across serotypes under various stress conditions

  • Assess interaction with host factors using infB from different serotypes

  • Evaluate differences in expression levels of both forms (alpha and beta) across serotypes

By implementing this comprehensive approach, researchers can account for serotype-specific differences while identifying conserved features of infB that could serve as universal targets for intervention strategies.

What are the methodological approaches for investigating potential interactions between infB and host cellular factors during infection?

Investigating interactions between S. pneumoniae infB and host cellular factors during infection requires sophisticated methodological approaches:

• Protein-protein interaction techniques:

  • Co-immunoprecipitation of infB with host proteins from infected cell lysates

  • Yeast two-hybrid screening to identify potential binding partners

  • Proximity labeling methods (BioID, APEX) to capture transient interactions in living cells

  • Surface plasmon resonance or biolayer interferometry to quantify binding kinetics

• Cell-based assays:

  • Fluorescence microscopy using tagged infB to track localization during infection

  • FRET/BRET approaches to monitor real-time interactions in living cells

  • Cell fractionation to determine compartmentalization of infB during infection

  • RNA-seq analysis of host cells exposed to purified infB to identify transcriptional responses

• In vivo approaches:

  • Mouse models with fluorescently tagged infB to track distribution during infection

  • Chromatin immunoprecipitation followed by sequencing (ChIP-seq) if infB interacts with host DNA

  • Immunohistochemistry of infected tissues to localize infB in relation to host factors

• Structural biology:

  • Cryo-EM or X-ray crystallography of infB-host protein complexes

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • In silico molecular docking to predict potential interaction sites

These methodologies can reveal whether infB functions beyond its canonical role in bacterial translation initiation by potentially modulating host cellular processes during infection.

How might differences in recombination rates across S. pneumoniae serotypes influence the evolution of infB and its potential as a therapeutic target?

The significant variation in recombination rates across S. pneumoniae serotypes has important implications for infB evolution and therapeutic targeting:

• Evolutionary considerations:

  • Serotypes with higher recombination rates (like serotype 6A with 8.91 recombination events per isolate) may show accelerated infB evolution compared to serotypes with lower rates (like serotype 1 with 0.73 events per isolate)

  • The positive association between capsule size and recombination suggests that serotypes with larger capsules may experience more rapid diversification of infB sequences

  • This differential evolution could lead to serotype-specific functional adaptations in infB

• Therapeutic targeting implications:

  • Higher recombination rates may facilitate more rapid escape from inhibitors targeting infB

  • Conserved regions of infB that remain invariant despite high recombination pressure represent ideal therapeutic targets

  • Developing inhibitors that target both alpha and beta forms could reduce the likelihood of resistance development

  • Combination therapies targeting multiple translation factors might be necessary for serotypes with high recombination rates

• Monitoring strategies:

  • Regular sequence surveillance of infB across serotypes to track evolutionary changes

  • Correlation of sequence changes with phenotypic properties like antimicrobial resistance

  • In vitro evolution experiments under drug pressure to predict resistance pathways

Understanding the relationship between recombination rates, capsule properties, and infB evolution is crucial for developing sustainable therapeutic strategies that remain effective despite S. pneumoniae's capacity for genetic adaptation .

What computational modeling approaches can help predict the functional impact of naturally occurring polymorphisms in the infB gene across S. pneumoniae lineages?

Advanced computational modeling approaches can provide valuable insights into the functional impact of naturally occurring infB polymorphisms across S. pneumoniae lineages:

These computational approaches, when integrated with experimental validation, provide a powerful framework for understanding how natural variation in infB contributes to functional diversity across pneumococcal lineages with different recombination rates, capsule sizes, and epidemiological characteristics .

What are the most promising future research directions for understanding infB's role in S. pneumoniae biology and pathogenesis?

Future research on S. pneumoniae infB should focus on several promising directions:

• Comparative functional studies of IF2 alpha and beta forms across different stress conditions and infection stages

• Investigation of serotype-specific variations in infB sequence and expression patterns

• Exploration of potential non-canonical functions beyond translation initiation

• Examination of infB's role in antibiotic resistance mechanisms

• Development of high-throughput screening methods for identifying infB inhibitors

• Integration of structural biology approaches to facilitate structure-based drug design

These directions would advance our understanding of this essential bacterial factor while potentially revealing new therapeutic opportunities for combating pneumococcal infections.

How might emerging technologies enhance our understanding of infB structure-function relationships?

Emerging technologies are poised to revolutionize our understanding of infB structure-function relationships:

• Cryo-electron microscopy advances allowing visualization of translation initiation complexes at near-atomic resolution

• Single-molecule techniques to observe infB dynamics during translation initiation in real-time

• CRISPR-based approaches for precise genome editing to study infB variants in native contexts

• Ribosome profiling to assess the global impact of infB variants on translation efficiency

• Systems biology approaches integrating multi-omics data to place infB function in broader cellular context