This study established the comprehensive repeating unit structure of immunologically active glucuronoxylomannan (AAPS) from wood ear mushroom, Auricularia auricula-judae. We identified Toll-like receptor 4 (TLR4) as a critical receptor involved in AAPS-induced macrophage activation to secrete pro-inflammatory cytokines. Molecular modeling data and chemical modifications of AAPS revealed that both carboxylic and acetyl moieties of AAPS are equally essential in TLR4 binding to exert in vitro immunostimulatory activity.

Traditional medicines provide a fertile ground to explore potent lead compounds, yet their transformation into modern drugs is fraught with challenges in deciphering the target that is mechanistically valid for its biological activity. Herein we reveal that (Z)-(+)-isochaihulactone (1) exhibited significant inhibition against multiple-drug-resistant (MDR) cancer cell lines and mice xenografts. NMR spectroscopy showed that 1 resisted an off-target thiolate, thus indicating that 1 was a target covalent inhibitor (TCI). By identifying the pharmacophore of 1 (α,β-unsaturated moiety), a probe derived from 1 was designed and synthesized for TCI-oriented activity-based proteome profiling. By MS/MS and computer-guided molecular biology approaches, an affinity-driven Michael addition of the noncatalytic C247 residue of GAPDH was found to control the "ON/OFF" switch of apoptosis through non-canonically nuclear GAPDH translocation, which bypasses the common apoptosis-resistant route of MDR cancers.

a. Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan b. Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan c. Key Laboratory of Molecular Biology of Heilongjiang Province, College of Life Sciences, Heilongjiang University, Harbin 150080, China d. Department of Chemistry, National Tsing-Hua University, Hsinchu, Taiwan e. Faculty of Applied Sciences, Sabaragamuwa University of Sri Lanka, Sri Lanka f. Department of Laboratory Medicine, Linsen, Chinese Medicine and Kunming Branch, Taipei City Hospital, Taipei, Taiwan g. Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan h. Department of Biotechnology and Animal Science, National Ilan University, Ilan, Taiwan

Concerns of Acinetobacter baumannii infection have increased due to the emergence of multi-drug resistance. In the present study, we determined the capsular polysaccharide (CPS) structure of A. baumannii SK44, a clinical isolate from Taiwan, to consist of pentasaccharide repeats. We found that CPS-induced antibody provided 55% protection against challenge in an animal model. The CPS-specific antibody reacted with the surface components of about 62% clinical isolates (342/554 strains) from cross-sectional and longitudinal studies by dot-immunoassay. Pulsed-field gel electrophoresis of positive strains showed the antibody covered different clonalites of A. baumannii clinical isolates. Meanwhile, using the CPS antibody as a probe, we found a number of outer membrane proteins bound to the antibody, including OmpA/motB, TonB-dependent receptor, and Omp38, indicating their association with CPS. These results might lead to the use of the capsular polysaccharide as a vaccine to prevent A. baumannii infection.

Nosocomial infectious outbreaks caused by multidrug-resistant Acinetobacter baumannii have emerged as a serious threat to human health. Phosphoproteomics of pathogenic bacteria has been used to identify the mechanisms of bacterial virulence and antimicrobial resistance. In this study, we used a shotgun strategy combined with high-accuracy mass spectrometry to analyze the phosphoproteomics of the imipenem-susceptible strain SK17-S and -resistant strain SK17-R. We identified 410 phosphosites on 248 unique phosphoproteins in SK17-S and 285 phosphosites on 211 unique phosphoproteins in SK17-R. The distributions of the Ser/Thr/Tyr/Asp/His phosphosites in SK17-S and SK17-R were 47.0%/27.6%/12.4%/8.0%/4.9% versus 41.4%/29.5%/17.5%/6.7%/4.9%, respectively. The Ser-90 phosphosite, located on the catalytic motif S88VS90K of the AmpC β-lactamase, was first identified in SK17-S. Based on site-directed mutagenesis, the nonphosphorylatable mutant S90A was found to be more resistant to imipenem, whereas the phosphorylation-simulated mutant S90D was sensitive to imipenem. Additionally, the S90A mutant protein exhibited higher β-lactamase activity and conferred greater bacterial protection against imipenem in SK17-S compared with the wild-type. In sum, our results revealed that in A. baumannii, Ser-90 phosphorylation of AmpC negatively regulates both β-lactamase activity and the ability to counteract the antibiotic effects of imipenem. These findings highlight the impact of phosphorylation-mediated regulation in antibiotic-resistant bacteria on future drug design and new therapies.

Discovery of new antibiotics for combating methicillin-resistant Staphylococcus aureus (MRSA) is of vital importance in the post-antibiotic era. Here, we report four avenaciolide derivatives (1-4) isolated from Neosartorya fischeri, three of which had significant antimicrobial activity against MRSA. The morphology of avenaciolide-treated cells was protoplast-like, which indicated that cell wall biosynthesis was interrupted. Comparing the structures and minimum inhibitory concentrations of 1-4, the α,β-unsaturated carbonyl group seems to be an indispensable moiety for antimicrobial activity. Based on a structural similarity survey of other inhibitors with the same moiety, we revealed that MurA was the drug target. This conclusion was validated by (31)P NMR spectroscopy and MS/MS analysis. Although fosfomycin, which is the only clinically used MurA-targeted antibiotic, is ineffective for treating bacteria harboring the catalytically important Cys-to-Asp mutation, avenaciolides 1 and 2 inhibited not only wild-type but also fosfomycin-resistant MurA in an unprecedented way. Molecular simulation revealed that 2 competitively perturbs the formation of the tetrahedral intermediate in MurA. Our findings demonstrated that 2 is a potent inhibitor of MRSA and fosfomycin-resistant MurA, laying the foundation for the development of new scaffolds for MurA-targeted antibiotics.