PURIFICATION AND BIOPHYSICAL CHARACTERIZATION OF PHOSPHOLIPASE-D FROM AUSTWICKIA CHELONAE FOR SEARCH FOR PEPTIDE INHIBITORS.

Austwickia chelonae is a gram-positive bacterium that causes dermatophilosis. This disease affects a wide range of vertebrates, including mammals, and is characterized by skin inflammation and epidermal necrosis. Different microorganisms, including A. chelonae, have phospholipase-D (PLD), an enzyme that cleaves phospholipids, altering the fluidity, permeability and charge of the cytoplasmic membrane. The pld gene of A. chelonae was first identified by sequencing the genome of this microorganism. Although this bacterium causes severe symptoms, stereochemical analyses to understand the mechanisms of action of its virulence factors are poorly elucidated. The primary alignment of the PLD of A. chelonae with other phospholipase-D from arachnids, bacteria and fungi indicated that the catalytic residues (H12 and H47) and coordination of Mg⁺² ions (E32, D34 and D91) are conserved, as well as its secondary structure. However, the catalytic activity of this toxin had not been tested in previous studies. For the first time, the protocol for the expression and purification of heterologous PLD from A. chelonae was established. Competent Escherichia coli BL21-(DE3) cells were transformed with the pET-28a(+) expression vector containing the PLD coding sequence. The preinoculum containing LB culture medium, kanamycin (50 mg/mL) and the bacteria was grown for 16 hours at 37 ºC and 180 rpm. The preinoculum was diluted one hundred times in a new LB medium also containing kanamycin (50 mg/mL). When the optical density at 600 nm reached 0.5, 0.4 mM IPTG was added to the cultures, which were maintained at 180 rpm and 30 ºC for 5 hours. Two purification steps were performed - affinity and molecular chromatography - the protein remained stable in PBS 1X buffer + 2 mM MgCl₂. The next step is to use A. chelonae PLD in phage display experiments to search for peptides that bind to secondary sites of the toxin. The identified peptides will first be evaluated by in silico assays and those with the best binding will be tested in in vitro experiments with HaCaT cells (human keratinocytes) and enzymatic assays. Furthermore, crystallization and biophysical characterization assays of PLD will be performed. 

This work is supported by FAPESP (2024/03185-6) and associated to FAPESP thematic project (2020/08615-8).