Recombinant Stenotrophomonas maltophilia 60 kDa chaperonin (groL), partial

What is Stenotrophomonas maltophilia 60 kDa chaperonin (groL) and what is its function?

The 60 kDa chaperonin (groL) of Stenotrophomonas maltophilia belongs to the chaperonin family of molecular chaperones found in many bacteria. It functions as a crucial protein folding machinery, assisting the proper folding of many proteins that would otherwise misfold or aggregate. The protein requires the co-chaperonin GroES to function properly, forming a barrel-like structure that creates an isolated environment for protein folding .

In eukaryotes, the mitochondrial heat shock protein 60 (Hsp60) is structurally and functionally similar to bacterial GroEL due to their endosymbiotic origin . The chaperonin system is essential for cellular viability, particularly under stress conditions when protein misfolding becomes more prevalent.

How does recombinant S. maltophilia groL expression differ from endogenous expression?

Recombinant S. maltophilia groL expression typically involves heterologous systems, most commonly E. coli. When expressing recombinant groL, researchers must consider several factors that differ from endogenous expression:

• Expression levels: Recombinant systems often produce higher concentrations than naturally occur in S. maltophilia

• Co-expression requirements: Optimal folding and function may require co-expression with GroES

• Post-translational modifications: These may differ between the native and recombinant systems

• Inclusion body formation: High-level expression often leads to protein aggregation requiring solubilization strategies

Studies have shown that co-expression of GroEL-GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli, potentially serving as a tool for enhanced production of multiple functional recombinant proteins .

What structural features define S. maltophilia groL and how do they relate to function?

The S. maltophilia groL protein shares the characteristic structure of other bacterial GroEL proteins, comprising:

• A double-ring tetradecameric structure (14 subunits)

• Three distinct domains: apical, intermediate, and equatorial

• An ATP binding pocket in the equatorial domain

• Hydrophobic residues in the apical domain for substrate binding

The functional cycle involves ATP binding, GroES association, substrate protein encapsulation, and conformational changes that facilitate proper folding. The large central cavity created by this structure provides an isolated environment that prevents aggregation of partially folded proteins.

How can recombinant S. maltophilia groL be utilized in experimental studies of bacterial pathogenesis?

Recombinant S. maltophilia groL can be employed to investigate several aspects of pathogenesis:

• Immunological studies: GroL proteins are often immunogenic and can be used to study host immune responses against S. maltophilia

• Protein-protein interaction analyses: Identifying host proteins that interact with S. maltophilia groL

• Stress response investigations: Examining how groL levels change during infection conditions

• Structure-function relationship studies: Using site-directed mutagenesis to identify residues critical for pathogenesis

The immunoproteomic approach has proven efficient for screening immunogenic proteins of S. maltophilia, which can lead to identification of potential vaccine candidates . Similar approaches could be used with groL to understand its role in host-pathogen interactions.

What is known about the role of groL in S. maltophilia antimicrobial resistance?

While the search results don't directly address groL's role in antimicrobial resistance, several hypotheses can be explored:

• GroL may help properly fold proteins involved in efflux pump assembly

• The chaperone could stabilize altered target proteins that would otherwise be destabilized by antibiotic binding

• Under antibiotic stress, increased groL expression might help maintain cellular proteostasis

S. maltophilia is known for its inherent antibiotic resistance , making it increasingly challenging to treat effectively. Understanding the role of groL in maintaining cellular functions under antibiotic pressure could provide insights into resistance mechanisms.

What experimental approaches can identify the client proteins of S. maltophilia groL?

Several methodological approaches can identify groL client proteins:

• Co-immunoprecipitation followed by mass spectrometry

• Bacterial two-hybrid screening

• Protein microarray analysis

• Comparative proteomics of wild-type vs. groL-depleted strains

• Cross-linking coupled with mass spectrometry

Table 1: Recommended experimental approaches for groL client protein identification

What are the optimal conditions for expressing and purifying recombinant S. maltophilia groL?

Expression and purification of functional recombinant S. maltophilia groL requires careful optimization:

Expression conditions:

• Host strain: BL21(DE3) or derivatives are typically preferred for chaperonin expression

• Growth temperature: 25-30°C often yields better solubility than 37°C

• Induction: Low IPTG concentrations (0.1-0.5 mM) with longer induction times

• Co-expression: Include GroES for improved folding and stability

Purification strategy:

• Cell lysis: Sonication or French press in buffer containing 20-50 mM Tris-HCl pH 7.5, 100-300 mM NaCl

• Initial capture: Affinity chromatography using His-tag or other suitable tags

• Intermediate purification: Ion exchange chromatography

• Polishing: Size exclusion chromatography to isolate properly assembled tetradecamers

Folding of aggregation-prone recombinant proteins through co-expression of chaperonin GroEL and GroES has become a popular practice to optimize preparation of functional proteins in E. coli .

How can researchers assess the functional activity of recombinant S. maltophilia groL?

Multiple assays can be employed to evaluate groL functionality:

• ATPase activity assay: Measures ATP hydrolysis rate using colorimetric phosphate detection

• Protein refolding assay: Monitors the refolding of denatured model substrates like malate dehydrogenase

• Aggregation prevention assay: Quantifies the ability of groL to prevent thermal aggregation of model proteins

• Co-chaperonin binding assay: Evaluates interaction with GroES using surface plasmon resonance

Table 2: Functional assays for recombinant S. maltophilia groL

Studies have demonstrated that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously, making them valuable tools for recombinant protein production .

What are the current challenges in developing S. maltophilia groL-based vaccines or therapeutics?

Several challenges exist in developing groL-based interventions:

• Cross-reactivity with human Hsp60, potentially causing autoimmune responses

• Identifying specific epitopes unique to S. maltophilia groL

• Demonstrating sufficient immunogenicity to confer protection

• Establishing appropriate animal models for testing efficacy

The immunoproteomic approach has proven efficient for screening immunogenic proteins of S. maltophilia . While current research has focused on outer membrane proteins like OmpA and Smlt4123 as vaccine candidates, similar approaches could potentially identify immunogenic epitopes in groL.

How does S. maltophilia groL contribute to bacterial survival under stress conditions?

As a molecular chaperone, groL likely plays critical roles in stress adaptation:

• Temperature stress: Preventing protein denaturation and aggregation at elevated temperatures

• Oxidative stress: Maintaining function of proteins damaged by reactive oxygen species

• Antibiotic stress: Preserving cellular proteostasis during antimicrobial treatment

• Nutrient limitation: Ensuring proper folding of key metabolic enzymes

S. maltophilia is an emerging global multi-drug-resistant organism that is increasingly challenging to treat effectively . Understanding how groL contributes to stress resistance could provide insights into bacterial persistence during infection and treatment.

What potential exists for using S. maltophilia groL as a diagnostic biomarker?

Recombinant S. maltophilia groL could serve as a diagnostic tool in several capacities:

• Serological detection of S. maltophilia infections through anti-groL antibody detection

• PCR-based identification using groL-specific primers

• Mass spectrometry-based identification in clinical samples

The growing importance of S. maltophilia as an opportunistic pathogen, particularly in cystic fibrosis patients , highlights the need for improved diagnostic approaches.