Recombinant Staphylococcus aureus Na (+)/H (+) antiporter subunit F1

What is the structural composition of the Staphylococcus aureus Na(+)/H(+) antiporter complex?

The S. aureus Na(+)/H(+) antiporter represents a novel type of multisubunit complex encoded by seven open reading frames (ORFs) that form a single operon. Sequence analysis revealed a promoter-like sequence upstream of the first ORF and a terminator-like sequence (inverted repeat followed by a T-cluster) downstream of the seventh ORF, with no terminator or promoter sequences between the ORFs . Hydropathy analysis indicates that all seven protein products are hydrophobic, consistent with their membrane-associated functions . This organization distinguishes the S. aureus antiporter from better-characterized systems like NhaA and NhaB in E. coli, which typically function as single-protein units.

How does the S. aureus Na(+)/H(+) antiporter differ from other bacterial antiporters?

The S. aureus Na(+)/H(+) antiporter exhibits several distinctive characteristics that set it apart from other bacterial Na(+)/H(+) antiporters:

• Multisubunit composition: Unlike the single-protein NhaA, NhaB, and ChaA antiporters of E. coli (which are typically encoded by ~1.5 kbp genes), the S. aureus system requires approximately 7 kbp of DNA encoding seven distinct subunits .

• pH activity profile: The S. aureus antiporter shows maximal activity at pH 7.0-7.5, unlike the NhaA antiporter from E. coli or V. parahaemolyticus (which functions optimally at alkaline pH 8.5) or the NhaB antiporter (which shows measurable activity at pH 7.0 but higher activity at pH 8.0-8.5) .

• Sequence characteristics: Homology searches have revealed that some subunits share sequence similarity with components of the respiratory chain, suggesting potential evolutionary relationships with other membrane transport systems .

What experimental systems are used to study recombinant S. aureus Na(+)/H(+) antiporter function?

The primary experimental system for studying the S. aureus Na(+)/H(+) antiporter involves heterologous expression in E. coli mutants lacking endogenous Na(+)/H(+) antiporters. Specifically:

• E. coli strain KNabc, which lacks all major Na(+)/H(+) antiporters, serves as an ideal host for functional characterization .

• Growth complementation assays in high-salt media (0.2 M NaCl or 10 mM LiCl) provide a functional readout, as KNabc cells cannot normally grow under these conditions, but those expressing the S. aureus antiporter can grow even in 0.8 M NaCl or 0.4 M LiCl .

• Na(+)/H(+) antiport activity is measured directly using everted membrane vesicles prepared from transformants, allowing quantification of Na(+) or Li(+) exchange for H(+) .

• Respiration-driven Na(+) extrusion can be measured to differentiate between direct Na(+) pumping and Na(+)/H(+) antiport mechanisms .

What are the recommended protocols for expressing and purifying recombinant S. aureus Na(+)/H(+) antiporter subunit F1?

While specific purification protocols for individual subunits are not detailed in the current literature, a general approach for recombinant expression of membrane proteins from S. aureus includes:

• Cloning strategy: The entire operon (approximately 7 kbp) should be cloned rather than individual subunits, as deletion analysis has shown that most of the operon is necessary for function . For individual subunit studies, co-expression with other subunits may be required for proper folding and stability.

• Expression system: E. coli KNabc strain provides an ideal background lacking competing antiporter activity . For purification purposes, addition of affinity tags (His6 or other epitope tags) to specific subunits like F1 should be carefully positioned to avoid disrupting protein function.

• Membrane protein extraction: Standard protocols using mild detergents (DDM, LDAO, or C12E8) that have been successful with other bacterial membrane transporters are recommended, with optimization required for the multisubunit complex.

• Functional verification: Antiporter activity should be confirmed in membrane vesicles before and after purification steps to ensure retention of function .

How does nitric oxide (NO·) stress affect the expression and function of S. aureus Na(+)/H(+) antiporter components?

The relationship between nitric oxide stress and S. aureus Na(+)/H(+) antiporter function represents an important area of current research:

• Transcriptional impacts: Tn-Seq studies have identified that genes encoding Na(+)/H(+) antiporters show altered fitness during NO· stress, suggesting regulatory links between nitrogen stress response and antiporter expression .

• Physiological importance: Since high concentrations of NO· inhibit normal respiration in S. aureus, the organism likely depends on secondary transporters, including Na(+)/H(+) antiporters, to maintain pH homeostasis and membrane potential under these conditions .

• Compensatory mechanisms: Research has established compensatory relationships between specific S. aureus antiporters in pH and proton regulation during non-NO· stress, which may extend to NO· stress conditions .

• Potential research approach: Quantitative PCR and western blot analysis of antiporter subunits (including F1) under controlled NO· exposure would help establish expression patterns, while membrane vesicle transport assays could determine functional impacts.

What structure-function relationships have been established for individual subunits of the S. aureus Na(+)/H(+) antiporter complex?

• Sequence homology: Some subunits show sequence similarity to components of the respiratory chain, suggesting potential evolutionary and functional relationships .

• Hydrophobicity profiles: All seven proteins are hydrophobic, consistent with membrane integration, though specific transmembrane topologies for individual subunits have not been fully mapped .

• Experimental approaches: Site-directed mutagenesis of conserved residues, particularly in predicted transmembrane regions, could help identify critical functional domains within each subunit, including F1.

• Cross-linking studies: Chemical cross-linking combined with mass spectrometry could help determine which subunits directly interact with each other and potentially identify the position of F1 within the complex.

What is the proposed mechanism for Na(+)/H(+) exchange in the multisubunit S. aureus antiporter compared to single-protein antiporters?

The mechanistic details of the S. aureus multisubunit antiporter remain to be fully elucidated, but several key observations provide insight:

• Complex coordination: The requirement for multiple subunits suggests a more complex ion translocation pathway than in single-protein antiporters like NhaA or NhaB.

• Stoichiometry: The Na(+):H(+) exchange ratio has not been definitively established for the S. aureus system, though standard antiporters typically operate with 1:1 or 1:2 stoichiometry.

• Respiratory chain connection: The sequence similarity between some antiporter subunits and respiratory chain components suggests potential integration with energy-generating systems .

• Research approach: Electrophysiological studies of the reconstituted complex in liposomes or planar lipid bilayers would help establish ion transport mechanisms and stoichiometry.

What role does the Na(+)/H(+) antiporter play in S. aureus pathogenesis and host colonization?

The Na(+)/H(+) antiporter likely contributes to S. aureus pathogenesis through several mechanisms:

• Survival under stress conditions: S. aureus is a halotolerant bacterium capable of surviving in 3 M NaCl or 1 M LiCl and can grow at pH values up to 9.5 . This remarkable salt and pH tolerance depends on Na(+)/H(+) antiporter activity, allowing the bacterium to persist in diverse host environments.

• Nitric oxide resistance: The antiporter system appears to contribute to S. aureus resistance against nitric oxide (NO·), a major component of host immune defense . This resistance mechanism has attracted scientific interest for potential drug targeting.

• Intracellular pH regulation: During host infection, S. aureus must maintain cytoplasmic pH homeostasis despite varying external conditions. The antiporter helps maintain a slightly alkaline cytoplasm optimal for bacterial function, even during NO· stress when respiration is inhibited .

• Research approach: Animal infection models comparing wild-type strains with antiporter mutants would help establish the in vivo relevance of this system to pathogenesis.

How can the S. aureus Na(+)/H(+) antiporter be targeted for antimicrobial development?

The multisubunit nature of the S. aureus Na(+)/H(+) antiporter presents both challenges and opportunities for antimicrobial development:

• Target validation: Tn-Seq studies have already identified antiporter genes as important for S. aureus fitness during NO· stress, providing preliminary target validation .

• Unique structure: The multisubunit organization differs significantly from human Na(+)/H(+) exchangers, potentially offering selectivity for antimicrobial targeting.

• Screening approaches: High-throughput screening using the E. coli KNabc complementation system provides a straightforward readout (growth in high salt) for identifying inhibitors .

• Combination strategies: Antiporter inhibitors might show synergy with existing antibiotics or immune components like NO· donors, enhancing bacterial clearance.

• Research direction: Structure-based drug design would benefit from detailed structural characterization of the entire complex or key subunits like F1.