Functional and expression characteristics identification of Phormicins, novel AMPs from Musca domestica with anti-MRSA biofilm activity, in response to different stimuli.

Antibiotic-resistant bacteria (including MRSA) in the clinic pose a growing threat to public health, and antimicrobial peptides (AMPs) have great potential as efficient treatment alternatives. Houseflies have evolved over long periods in complex, dirty environments, developing a special immune system to overcome challenges in harmful environments. AMPs are key innate immune molecules. Herein, two differentially expressed AMPs, Phormicins A and B, were identified by screening transcriptomic changes in response to microbial stimulation. Structural mimic assays indicated that these AMPs exhibited functional divergence due to their C-terminal features. Expression analysis showed that they had different expression patterns. Phormicin B had higher constitutive expression than Phormicin A. However, Phormicin B was sharply downregulated, whereas Phormicin A was highly upregulated, after microbial stimulation. The MIC, MBC and time-growth curves showed the antibacterial spectrum of these peptides. Crystal violet staining and SEM showed that Phormicin D inhibited MRSA biofilm formation. TEM suggested that Phormicin D disrupted the MRSA cell membrane. Furthermore, Phormicin D inhibited biofilm formation by downregulating the expression of biofilm-related genes, including altE and embp. Therefore, housefly Phormicins were functionally characterized as having differential expression patterns and antibacterial & antibiofilm activities. This study provides a new potential peptide for clinical MRSA therapy.

Lactobacillus brevis CGMCC 1306 glutamate decarboxylase: Crystal structure and functional analysis.

Glutamate decarboxylase (GAD), which is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme, can catalyze α-decarboxylation of l-glutamate (L-Glu) to γ-aminobutyrate (GABA). The crystal structure of GAD in complex with PLP from Lactobacillus brevis CGMCC 1306 was successfully solved by molecular-replacement, and refined at 2.2 Šresolution to an Rwork factor of 18.76% (Rfree = 23.08%). The coenzyme pyridoxal 5-phosphate (PLP) forms a Schiff base with the active-site residue Lys279 by continuous electron density map, which is critical for catalysis by PLP-dependent decarboxylase. Gel filtration showed that the active (pH 4.8) and inactive (pH 7.0) forms of GAD are all dimer. The residues (Ser126, Ser127, Cys168, Ile211, Ser276, His278 and Ser321) play important roles in anchoring PLP cofactor inside the active site and supporting its catalytic reactivity. The mutant T215A around the putative substrate pocket displayed an 1.6-fold improvement in catalytic efficiency (kcat/Km) compared to the wild-type enzyme (1.227 mM-1 S-1 versus 0.777 mM-1 S-1), which was the highest activity among all variants tested. The flexible loop (Tyr308-Glu312), which is positioned near the substrate-binding site, is involved in the catalytic reaction, and the conserved residue Tyr308 plays a vital role in decarboxylation of L-Glu.