Biology subjects

Bosetto, F.

Publications and source records attributed to Bosetto, F..

3 recordsLinked to original sources

bioRxiv2026

DNA aptamers to interfere with HasA, the hemophore of the heme assimilation system in Pseudomonas aeruginosa, as a potential antimicrobial strategy

Pseudomonas aeruginosa relies on heme acquisition to sustain growth and virulence in the iron-limited environment of the host, particularly in the acidic airways of patients with cystic fibrosis where antibiotic efficacy is markedly reduced. The Has heme assimilation system is initiated by the secreted hemophore HasA, which binds extracellular heme and delivers it to the outer-membrane receptor HasR. During infection, HasA is proteolytically processed, generating a truncated form that constitutes the biologically relevant species. Here, we characterise the structural and functional properties of truncated HasA under disease-associated acidic conditions (pH 6.5) and perform a rationally designed Systemic Evolution of Ligands by EXponential enrichment (SELEX) method to identify DNA aptamers capable of binding this hemophore. Biophysical analyses revealed that truncated HasA is folded, displays a mixed /{beta} secondary structure, and exists as a concentration-dependent mixture of monomers and domain-swapped dimers. Circular dichroism and nano-differential scanning fluorimetry identified two thermal transitions, consistent with the coexistence of apo/holo and monomer/dimer species. The apo protein bound heme with high affinity (KD = 113 nM) and a 1:1 stoichiometry, confirming preservation of its functional binding mechanism. Twelve rounds of SELEX, incorporating target switching and platform switching, yielded a highly enriched aptamer pool dominated by two sequences, HasA_1 and HasA_2. ELONA (Enzyme-Linked Oligonucleotide Assay) assays demonstrated that both aptamers bind specifically to truncated HasA, and biolayer interferometry revealed nanomolar dissociation constants (KD = 0.59 M and 0.32 M, respectively). These aptamers did not bind full-length HasA, BSA, or control sequences, confirming that selection under acidic conditions drove specificity toward the physiologically relevant form of the hemophore. Our findings identify HasA-binding DNA aptamers that retain function in acidic environments where antibiotic potency is compromised, highlighting their potential as molecular tools to disrupt heme acquisition in P. aeruginosa. This work establishes a foundation for developing aptamer-based antimicrobial strategies targeting the Has system, particularly under acidic conditions where conventional antibiotics are compromised.

biophysics↗

bioRxiv2026

A generalisable method for the purification and biophysical characterisation of bacterial membrane receptors

Membrane-embedded bacterial receptors are challenging to express and purify in soluble form, yet their isolated domains are essential tools for structural and ligand-discovery studies. Pseudomonas aeruginosa relies on the TonB-dependent heme receptor HasR for iron acquisition, a process central to its pathogenicity. Here, we report a robust strategy for the recombinant expression, purification, and biophysical characterisation of the two soluble HasR domains directly involved in heme uptake: the N-terminal plug and the Secretin/TonB short N-terminal domain. Each domain was expressed individually in E. coli and purified to homogeneity, adopting well-folded conformations as confirmed by circular dichroism, NMR spectroscopy, and mass spectrometry. We then engineered a fusion construct containing both domains and systematically evaluated multiple solubilisation tags. A GST-His dual-affinity strategy enabled efficient purification of the construct, whereas His-tag alone resulted in insoluble protein and HLT-tag fusions suffered from non-specific proteolysis. Biophysical analyses revealed that the Secretin/TonB short N-terminal domain remains stably folded within the fusion construct, while the N-terminal plug domain becomes partially disordered, a finding further supported by hydrogen/deuterium exchange mass spectrometry. Together, these results establish a generalizable workflow for producing soluble receptor domains from membrane proteins and provide validated HasR constructs suitable for downstream ligand-screening applications, including aptamer and nanobody discovery.

biophysics↗

bioRxiv2021

Knocking out AtCALS7 disturbs photoassimilate distribution and weakens defence against phytoplasma infection

Callose deposition around sieve pores, under control of callose synthase 7 (AtCALS7), has been interpreted as a mechanical response to limit pathogen spread in phytoplasma-infected plants. Wild-type and Atcals7ko mutants were therefore employed to unveil the mode of involvement of CALS7 in the plants response to phytoplasma infection. The fresh weights of healthy and CY-(Chrysanthemum Yellows) phytoplasma-infected Arabidopsis wild-type and mutant plants indicated two superimposed effects of the absence of CALS7: a partial impairment of photo-assimilate transport and a stimulated phytoplasma proliferation as illustrated by a significantly increased phytoplasma titre in Atcal7ko mutants. Further studies solely dealt with the effects of CALS7 absence on phytoplasma growth. Phytoplasma infection affected sieve-element substructure to a larger extent in mutants than in wild-type plants, which was also true for the levels of some free carbohydrates. Moreover, infection induced a similar upregulation of gene expression of enzymes involved in sucrose cleavage (AtSUS5, AtSUS6) and transmembrane transport (AtSWEET11) in mutants and wild-type plants, but an increased gene expression of carbohydrate transmembrane transporters (AtSWEET12, AtSTP13, AtSUC3) in infected mutants only. It remains still unclear how the absence of AtCALS7 leads to gene upregulation and how an increased intercellular mobility of carbohydrates and possibly effectors contributes to a higher susceptibility. It is also unclear if modified sieve-pore structures in mutants allow a better spread of phytoplasmas giving rise to higher titre. Author SummaryPhytoplasma infections are one of the most limiting factors for production of important crops all over the world. Phytoplasma disease epidemics can be handled mainly by insect-vector control using insecticides. Basic information about plant-phytoplasma interactions are still limited, nevertheless it is necessary to design new management and breeding strategies aimed to obtain more tolerant or resistant cultivars. Phytoplasmas are obligate intracellular parasites restricted to the phloem sieve tubes. Callose deposition at the sieve plates has been described since the 70s as a mechanical defence process to limit pathogen spread by occluding sieve pores. Studies reported 40 years later demonstrated that callose at the sieve pores is also involved in sieve-pore development and function and, hence, in mass-flow regulation, carbohydrate metabolism and distribution, and plant growth. Here, we reported on the role(s) of sieve-element callose in phytoplasma-infected Arabidopsis, using a mutant lacking AtCALS7, the enzyme responsible for callose synthesis in the sieve elements. The results indicate that loss of AtCAL7 appears to confer increased susceptibility to phytoplasma infection, due to alterations in expression of genes involved in sugar metabolism and membrane transport. In the long run, the identification of plant resistance or susceptibility traits against phytoplasmas will allow a complete re-organization of chemical control strategies, with obvious opportunities of reducing insecticide burden.

plant biology↗