Pseudomonas aeruginosa uses extracellular heme uptake to support growth and virulence in iron-limited host niches. The Has heme assimilation system is initiated by the secreted hemophore HasA, which is proteolytically processed during infection to a truncated form that represents the biologically relevant species. The study characterized this truncated HasA under disease-associated acidic conditions (pH 6.5).
Biophysical analyses showed that truncated HasA is folded and presents a mixed α/β secondary structure. The protein exists as a concentration-dependent mixture of monomers and domain-swapped dimers, indicating a dynamic oligomeric equilibrium in solution. The presence of domain-swapped dimers suggests structural plasticity that may be relevant for function or interaction with ligands under physiological conditions.
Circular dichroism (CD) and nano-differential scanning fluorimetry (nano-DSF) experiments identified two distinct thermal transitions for truncated HasA. The authors interpreted these transitions as consistent with coexistence of different molecular populations, specifically apo versus holo forms and monomer versus dimer species.
Functional heme binding was retained: the apo truncated HasA bound heme with high affinity, with a reported dissociation constant (KD) of 113 nM and a 1:1 stoichiometry. This result confirms that the truncated species preserves the canonical heme-binding mechanism of the hemophore despite proteolytic processing and the acidic test conditions.
To discover nucleic acid ligands capable of binding the physiologically relevant hemophore, the investigators implemented a rationally designed SELEX (Systematic Evolution of Ligands by EXponential enrichment) workflow. Selection was performed under acidic conditions (pH 6.5) to mirror disease-relevant environments where P. aeruginosa relies on heme uptake and where antibiotic efficacy can be compromised.
The SELEX protocol included twelve rounds of enrichment and incorporated both target switching and platform switching steps. These deliberate variations in the selection process aimed to increase the likelihood of isolating aptamers that recognize structural features specific to the truncated form of HasA and that remain functional under acidic conditions.
After twelve rounds of SELEX, the enriched aptamer pool was dominated by two sequences, designated HasA_1 and HasA_2. The deep enrichment of these sequences indicates strong selective pressure in the SELEX protocol for ligands that bind truncated HasA at pH 6.5.
The study therefore identified candidate DNA aptamers that preferentially interact with the physiologically relevant hemophore form produced during infection.
Binding of the selected aptamers to truncated HasA was validated using ELONA (Enzyme-Linked Oligonucleotide Assay). Both HasA_1 and HasA_2 showed specific binding to truncated HasA in these assays.
Quantitative kinetics were assessed by biolayer interferometry (BLI), which produced dissociation constants in the submicromolar to low-micromolar range: KD = 0.59 μM for HasA_1 and KD = 0.32 μM for HasA_2. These BLI-derived affinities indicate a high degree of binding under the experimental conditions and support the biochemical relevance of the selected aptamers.
Specificity controls showed that HasA_1 and HasA_2 did not bind full-length HasA, bovine serum albumin (BSA), or control sequences. This lack of cross-reactivity indicates that selection under acidic conditions successfully directed specificity toward the truncated, infection-relevant form of the hemophore rather than the full-length secreted protein or unrelated proteins.
By identifying aptamers that retain function at pH 6.5, the work addresses a clinically relevant environment where conventional antibiotics often lose potency—most notably the acidic airways of people with cystic fibrosis. The aptamers’ specificity for truncated HasA suggests they could be used as molecular tools to disrupt the initial step of heme acquisition by P. aeruginosa under pathophysiological conditions.
The study establishes a foundation for developing aptamer-based antimicrobial strategies that target the Has system in P. aeruginosa. Identified DNA aptamers bind the physiologically relevant truncated hemophore with nanomolar-to-micromolar affinity and do so under acidic conditions where antibiotic efficacy is compromised.
These findings support the potential utility of aptamers as targeted inhibitors or molecular probes to interfere with heme uptake, an essential nutrient pathway for P. aeruginosa during infection. The source did not report in vivo efficacy, therapeutic delivery approaches, or clinical testing; those details were not reported in the article. Further development would require follow-up studies to assess functional inhibition of heme acquisition in bacterial cultures and animal models, stability and delivery in relevant tissues, and safety profiles.
Competing interests and funding
The authors declared no competing interests. Funding sources and fellowships supporting the work were reported but detailed grant mechanisms and downstream translational plans were not described in the source.