Recombinant Mycobacterium abscessus Translation initiation factor IF-2 (infB), partial

What is Mycobacterium abscessus and why is it clinically important?

Mycobacterium abscessus is a rapid growing mycobacterium (RGM) that has emerged as a difficult-to-treat pathogen, especially in cystic fibrosis (CF) patients. It can cause pulmonary disease in immunocompetent individuals and shares characteristics with M. tuberculosis, including the ability to induce granulomatous lesions or caseous necrosis . M. abscessus is notorious for being highly drug-resistant to antimicrobial agents, making infections challenging to treat . The incidence of M. abscessus infections has been increasing worldwide, and in South Korea, it accounts for 70-80% of lung diseases caused by RGM .

What is Translation Initiation Factor IF-2 (infB) and what is its role in bacterial cells?

Translation Initiation Factor IF-2 is a key protein involved in the initiation of protein synthesis. Based on studies in E. coli, infB interacts with at least three components during translation initiation: GTP, fMet-tRNA (formylmethionyl-transfer RNA), and ribosomes . It initiates the binding of fMet-tRNA to the 70S ribosome in a process involving hydrolysis of GTP to GDP, thus playing a crucial role in the correct initiation of protein synthesis . The infB gene codes for multiple forms of the IF-2 protein, with IF2α (97,300 daltons) and IF2β (79,700 daltons) being the primary variants in E. coli .

How is the infB gene organized in bacterial genomes?

In E. coli, the infB gene contains internal in-frame initiation sites that allow for the expression of multiple forms of IF-2. The gene codes for the full-length IF2α (890 amino acids) as well as shorter forms like IF2β and IF2γ, which lack the N-terminal portions of the protein . A study of the infB gene in clinical isolates of E. coli found it to be extremely conserved, with only one polymorphic position in the deduced 890 amino acid sequence, located within the central GTP/GDP-binding domain IV of IF2 . This high conservation suggests a critical functional role for this protein in bacterial survival and growth.

What are the structural domains of bacterial IF-2 proteins?

Based on E. coli studies, IF-2 has a six-domain structural model. Domain I constitutes the difference between IF2α and IF2β forms. The GTP-binding domain is located within domain IV, and the C-terminal domain VI is involved in tRNA binding . The N-terminal domains (I, II, and III) have been found to be extremely conserved in E. coli clinical isolates, indicating a specific functional importance of this region . While the specific structure of M. abscessus IF-2 has not been extensively characterized, it likely shares similar domain organization with other bacterial IF-2 proteins.

How do the different forms of IF-2 (α, β, γ) differ functionally?

In E. coli, the two major forms of IF-2 (IF2α and IF2β) differ at their N-terminus. N-terminal sequencing of purified IF2α and IF2β revealed completely different N-terminal amino acid sequences that match the DNA sequences at the beginning of the infB open reading frame and an in-phase region 471 bp downstream . These differences at the N-terminus likely affect how the protein interacts with other components of the translation machinery. Both forms retain the ability to initiate protein synthesis but may have specialized roles in different cellular contexts or under different growth conditions.

What experimental evidence supports the independent translation of multiple IF-2 forms?

A fusion construct between the proximal half of the infB gene and the lacZ gene lacking the region coding for the first eight amino acids was created to study IF-2 forms. This fused gene expressed two products of 170,000 and 150,000 daltons, corresponding to IF2α-beta-galactosidase and IF2β-beta-galactosidase fusion proteins . Deletion of the 5'-non-translated region of the fused gene, including the Shine/Dalgarno ribosomal binding site, resulted in the expression of IF2β-beta-galactosidase but not IF2α-beta-galactosidase. This strongly suggests that IF2β results from independent translation rather than from proteolytic cleavage of IF2α .

How might IF-2 function differently in M. abscessus compared to other bacterial species?

M. abscessus has unique biological characteristics, including the ability to transition between smooth (S) and rough (R) morphotypes, which significantly affects its virulence and host immune response . This morphological transition is associated with exacerbation of disease and persistence of M. abscessus in patients . Given these unique characteristics, it's possible that IF-2 in M. abscessus has adapted specific functional attributes to support protein synthesis under the challenging conditions of host infection and antibiotic stress. Comparative studies of IF-2 across mycobacterial species would be needed to identify these potential adaptations.

Is there evidence for IF-2 involvement in M. abscessus antibiotic resistance?

M. abscessus is known for being one of the most highly drug-resistant mycobacterial species . While direct evidence linking IF-2 to antibiotic resistance in M. abscessus is lacking in the current literature, translation initiation factors could potentially contribute to resistance mechanisms. Some antibiotics target components of the translation machinery, and alterations in translation factors could affect susceptibility to these drugs. Additionally, IF-2's role in initiating protein synthesis could influence the expression of proteins involved in antibiotic resistance, such as efflux pumps or enzymes that modify antibiotics.

How does the stringent response in M. abscessus potentially affect IF-2 function?

Studies have shown that in M. abscessus, the stringent factor Rel regulates metabolism during stress conditions . The stringent response is a conserved signaling system that promotes bacterial survival under stress by altering the transcription of about a quarter of the genome . While the specific impact on IF-2 is not documented, the stringent response typically downregulates processes related to active growth, including protein synthesis. This suggests that IF-2 activity might be modulated during stress conditions as part of the broader physiological adaptation mediated by the stringent response.

What are the recommended strategies for cloning and expressing the M. abscessus infB gene?

For cloning the M. abscessus infB gene, researchers should consider:

• Designing PCR primers based on the published M. abscessus genome sequence, incorporating appropriate restriction sites for subsequent cloning

• Using high-fidelity DNA polymerase due to the large size of the gene (~2.7 kb based on E. coli infB )

• Optimizing PCR conditions for GC-rich mycobacterial DNA

• Cloning into expression vectors with inducible promoters

For expression:

• Consider E. coli strains specialized for expressing mycobacterial proteins

• Optimize expression conditions (temperature, inducer concentration, duration)

• Use fusion tags that enhance solubility (MBP, GST, SUMO) if initial expression yields insoluble protein

• Consider codon optimization if expression levels are low

What purification methods are most effective for recombinant M. abscessus IF-2?

Based on general protein purification principles and experience with similar proteins:

• Affinity chromatography using His-tag or other fusion tags is typically the first step

• Ion-exchange chromatography can separate different charged species

• Size exclusion chromatography helps remove aggregates and ensures homogeneity

• For functional studies, consider removing tags using specific proteases

• Optimize buffer conditions (pH, salt concentration, glycerol) to maintain stability during purification

Recombinant IF-2 proteins are typically purified in the presence of reducing agents to prevent oxidation of cysteine residues, and protease inhibitors to prevent degradation during the purification process.

What are the optimal storage conditions for recombinant M. abscessus IF-2?

Based on information from commercial recombinant infB products and general protein storage principles:

• Store at -20°C/-80°C for long-term preservation

• Add 5-50% glycerol as a cryoprotectant (50% is commonly used)

• Aliquot to avoid repeated freeze-thaw cycles

• The typical shelf life of liquid form is approximately 6 months at -20°C/-80°C

• The shelf life of lyophilized form is approximately 12 months at -20°C/-80°C

• Short-term storage at 4°C is limited to about one week

Working concentrations typically range from 0.1-1.0 mg/mL in appropriate buffer systems .

How can researchers assess the translation initiation activity of recombinant M. abscessus IF-2?

Several methods can be employed to assess the functional activity of recombinant IF-2:

• In vitro translation assays: Using purified components of the translation machinery to measure the ability of IF-2 to initiate protein synthesis

• GTP binding and hydrolysis assays: Measuring the binding of radiolabeled or fluorescent GTP analogs and the rate of GTP hydrolysis

• Ribosome binding assays: Assessing the interaction between IF-2 and purified ribosomes using techniques like surface plasmon resonance or filter binding assays

• fMet-tRNA binding assays: Measuring the ability of IF-2 to bind formylated initiator tRNA

• Dipeptide synthesis assay: An in vitro assay containing fMet-tRNA and various labeled aminoacyl-tRNAs to assess the formation of the first peptide bond, which requires functional IF-2

What assays can be used to study the interaction of IF-2 with other components of the translation machinery?

Researchers can study IF-2 interactions using:

• Pull-down assays with tagged IF-2 to identify interacting partners

• Biolayer interferometry or surface plasmon resonance to measure binding kinetics

• Fluorescence anisotropy to study interactions with fluorescently labeled ligands

• Isothermal titration calorimetry to determine thermodynamic parameters of binding

• Cross-linking studies followed by mass spectrometry to identify interaction sites

• Cryo-electron microscopy to visualize IF-2 in complex with ribosomes and other translation components

How can researchers investigate the role of IF-2 in M. abscessus morphotype transitions?

To study IF-2's potential role in morphotype transitions, researchers could:

• Compare IF-2 expression levels in smooth (S) and rough (R) variants using quantitative PCR or western blotting

• Create IF-2 knockdown strains using antisense RNA or CRISPR interference to assess impact on morphotype stability

• Perform RNA-seq analysis of S and R variants to identify changes in IF-2-dependent gene expression

• Use chromatin immunoprecipitation followed by sequencing (ChIP-seq) if IF-2 has potential regulatory roles beyond translation

• Create reporter strains to monitor IF-2 expression during S to R transition, which occurs in patients with exacerbation of disease and persistence of M. abscessus

How can recombinant M. abscessus IF-2 be used to develop new therapeutic approaches?

Recombinant M. abscessus IF-2 could contribute to therapeutic development through:

• Structure-based drug design: Solving the crystal structure of M. abscessus IF-2 to identify potential binding pockets for small molecule inhibitors

• High-throughput screening: Using purified IF-2 in functional assays to screen compound libraries for inhibitors

• Rational design of peptide inhibitors that disrupt IF-2 interactions with other components of the translation machinery

• Development of antibodies or aptamers that specifically target M. abscessus IF-2

• Investigation of synergistic effects between IF-2 inhibitors and existing antibiotics, particularly important since M. abscessus infections are notoriously difficult to treat due to antimicrobial resistance

What role might IF-2 play in the host immune response to M. abscessus infection?

The transition from smooth (S) to rough (R) morphotype in M. abscessus significantly affects the host immune response . While IF-2's direct role in immune response modulation is unknown, as a central player in protein synthesis, it could influence:

• Expression of bacterial surface components that interact with host immune receptors

• Production of virulence factors that modulate host immunity

• Adaptation to stress conditions imposed by host immune responses

The R morphotype of M. abscessus activates a stronger TLR2-mediated inflammatory response compared to the S morphotype . Studies have shown that infection with an R variant causes an inflammatory immune response that drives necrotic granuloma formation through host TNF signaling . Understanding how IF-2 functions during these different infection stages could provide insights into virulence mechanisms.

How might comparative analysis of IF-2 across mycobacterial species inform M. abscessus research?

Comparative analysis of IF-2 across mycobacterial species could:

• Identify unique structural features of M. abscessus IF-2 that could be exploited for species-specific targeting

• Reveal evolutionary adaptations in IF-2 that correlate with pathogenicity or drug resistance

• Provide insights into whether IF-2 variants contribute to the different clinical presentations of various mycobacterial infections

• Guide the rational design of broad-spectrum or species-specific translation inhibitors

Such comparative studies would be particularly valuable given the increasing prevalence of M. abscessus infections and the challenges in treating them compared to other mycobacterial diseases .

What are the main technical challenges in working with recombinant M. abscessus IF-2?

Researchers face several challenges when working with recombinant M. abscessus IF-2:

• Protein size: IF-2 is a large protein (approximately 97 kDa for the α form in E. coli ), which can complicate expression and purification

• Solubility: Large multi-domain proteins often have solubility issues when expressed recombinantly

• Functional activity: Ensuring that the recombinant protein retains its native activity after purification

• Multiple forms: The presence of multiple natural forms (α, β, γ) complicates expression and functional studies

• Domain interactions: The multi-domain structure may result in complex folding requirements

What are promising future research directions for understanding IF-2 function in M. abscessus?

Future research directions could include:

• Determining the crystal or cryo-EM structure of M. abscessus IF-2 in various functional states

• Investigating the role of IF-2 in antibiotic tolerance mechanisms

• Exploring the relationship between the stringent response and IF-2 function in M. abscessus

• Developing IF-2 inhibitors as potential adjuvants to current antibiotic regimens

• Creating conditional knockdown strains to study the essentiality of IF-2

• Investigating the potential role of IF-2 in the formation of antibiotic-tolerant persister cells

How might understanding IF-2 contribute to overcoming M. abscessus treatment challenges?

M. abscessus infections are challenging to treat due to intrinsic and acquired antimicrobial resistance . Understanding IF-2 could contribute to treatment advances by:

• Providing a new target for antimicrobial development

• Identifying mechanisms of translational adaptation during antibiotic stress

• Understanding how protein synthesis is regulated during infection

• Developing biomarkers based on IF-2 activity or expression to monitor treatment efficacy

• Creating diagnostic tools based on IF-2 sequence or activity to identify M. abscessus subspecies with different treatment outcomes (e.g., M. abscessus subsp. massiliense vs. M. abscessus subsp. abscessus, which show different treatment responses to clarithromycin-containing regimens )

Comparison of IF-2 characteristics across bacterial species

Recommended storage and handling conditions for recombinant IF-2 proteins

abscessus strain characteristics relevant to translation machinery studies