To investigate the interaction between a novel antimicrobial compound, HL-J6, and penicillin-binding protein 1 (PBP1) of Staphylococcus aureus.

With MRSA252 genomic DNA as the template and PBP1F and PBP1R as primers, the expression plasmid pET30a-pbp1-39-608 was constructed by amplifying the target gene fragment followed by cloning into the Nde Ⅰ/Xho Ⅰ restriction sites of the pET30a vector. Then the obtained plasmids were transformed into Escherichia coli for the expression of PBP1-39-608 protein, and the product was purified by affinity chromatography. The inhibitory effect of HL-J6 on the transpeptidase activity of PBP1-39-608 was measured using peptidoglycan side chain backbone peptide, with thiol ester analog S2d as the substrate. The affinity between HL-J6 and PBP1-39-608 was detected using microscale thermophoresis (MST), and the binding interaction was confirmed by cellular thermal shift assay (CETSA). Molecular docking and dynamics simulation were performed using AutoDock Vina and Desmond software, respectively, to elucidate the binding mode of HL-J6 with the PBP1-39-608 protein and the key amino acid residues involved.

The recombinant plasmid pET30a-pbp1-39-608 was successfully constructed, and PBP1-39-608 protein was produced after induction and purified, yielding a protein with an approximate molecular mass of 65×10³. HL-J6 inhibited the transpeptidase activity of PBP1-39-608 in a time-dependent manner (P<0.001). The dissociation constant Kd of the binding between HL-J6 and PBP1-39-608 was 64.92 μ mol/L. Molecular docking results showed that HL-J6 bound to the active pocket of PBP1-39-608 by interacting with key residues such as ILE-348, ASN-370, THR-516 and PHE-423, with a binding score of-8.38 kcal/mol (<-5.00 kcal/mol). Dynamics simulation results indicated that the complex became stable after 50 ns.

HL-J6 effectively inhibits the transpeptidase activity of Staphylococcus aureus PBP1, and shows stable interaction with the protein.

To compare the immune effects of recombinant PKF protein of Acinetobacter baumannii with different immune modes and different adjuvants.

After recombinant PKF protein was obtained by plasmid expression and purification, the protein was then combined with aluminum hydroxide（Al）or with AS03 to immunize mice by intramuscular（im）injection. In another experiment, the combination of PKF and LTK63 or PKF and LP1-34 was adopted to intranasally（in）immunize mice, respectively. The total animals were thus divided into 7 groups: PBS group; PKF group（im）, PKF+Al group（im）, PKF+AS03 group（im）, PKF group（in）, PKF+LTK63 group（in）, and PKF+LP1-34 group（in）. The levels of serum specific IgG and mucosal sIgA in the immunized mice were detected by ELISA. Moreover, the sublethal model of Acinetobacter baumannii pulmonary infection was established, and then the bacterial colonization amount in blood and lung of mice after challenge was measured to evaluate the protective immune efficacy of the recombinant vaccine.

In the intramuscular immunization group, AS03-assisted PKF produced the highest specific IgG antibody level（P<0.01）, and in the nasal immunization group, LTK63 significantly increased the level of specific sIgA in the alveolar lavage fluid（P<0.01）. Bacterial challenge test showed that the bacterial colonization amount was similar in the PKF+AS03（im）group and PKF（im）alone group, while the PKF+LTK63 group（in）obtained remarkably reduced bacterial colonization amount（P<0.01）.

LTK63 assists PKF to better protect against Acinetobacter baumannii pulmonary infection, suggesting that vaccine-mediated mucosal immune response may play a more important role in the infection protection.