Development of non-ß-Lactam covalent allosteric inhibitors targeting PBP2a in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive bacterial pathogen, continues to pose a serious threat to the current public health system in our society. The high level of resistance to ß-lactam antibiotics in MRSA is attributed to the expression of penicillin-binding protein 2a (PBP2a), which catalyzes cell wall cross-linking. According to numerous research reports, the activity of the PBP2a protein is known to be regulated by an allosteric site distinct from the active site where cell wall cross-linking occurs. Here, we conducted a screening of 113 compounds containing a 1,3,4-oxadiazole core to design new covalent inhibitors targeting the allosteric site of PBP2a and establish their structural-activity relationship. The stereochemically selective synthesis of sulfonyl oxadiazole compounds identified in the initial screening resulted in a maximum eightfold enhancement in cell inhibition activity. The sulfonyl oxadiazole-based compounds formulated as PEG-based ointments, with low toxicity test results on human cells (CC 50 : >78µM), demonstrated potent antimicrobial effects not only in a mouse skin wound infection model but also against oxacillin-resistant clinical isolate MRSA (IC 50 ≈ 1µM), as evidenced by the results. Furthermore, additional studies utilizing LC-MS/MS and in-silico approaches clearly support the allosteric site covalent binding mechanism through the nucleophilic aromatic substitution (S N Ar) reaction, as well as its association with the closure of the major active site of PBP2a.

Carbapenem-induced ß-lactamase-isoform expression trends in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an urgent bacterial threat to public health, with only a few treatment options and a >50% fatality rate. Although several resistance mechanisms are understood, the appearance of these mutations is generally considered stochastic. Recent reports have, however, begun to challenge this assumption. Here, we demonstrate that independent samples of Ab, exposed to different carbapenems with escalating concentrations, show concentration- and carbapenem-dependent trends in ß-lactamase-isoform expression. This result, based on the isoforms identified through label-free-quantification LC-MS/MS measurements of cell-free, gel-separated ß-lactamases, suggests that the appearance of antibiotic resistance may be somewhat non-stochastic. Specifically, several minor AmpC/ADC ß-lactamase-isoforms were found to exhibit both dose- and carbapenem-dependent expression, suggesting the possibility of non-stochastic mutations. Additionally, these also have high sequence similarity to major expressed isoforms, indicating a potential path over which resistance occurred in independent samples. Antibiotic resistance maybe somewhat antibiotic-directed by a hitherto unknown mechanism and further investigation may lead to new strategies for mitigating antibiotic resistance. Teaser: The emergence of antibiotic-resistant ß-lactamase proteins from mutations may exhibit patterns based on specific antibiotics.

Effect of aerobic exercise and particulate matter exposure duration on the diversity of gut microbiota.

Inhalation of ambient particulate matter (PM) can disrupt the gut microbiome, while exercise independently influences the gut microbiome by promoting beneficial bacteria. In this study, we analyzed changes in gut microbial diversity and composition in response to combined interventions of PM exposure and aerobic exercise, extending up to 12 weeks. This investigation was conducted using mice, categorized into five groups: control group (Con), exercise group (EXE), exercise group followed by 3-day exposure to PM (EXE + 3-day PM), particulate matter exposure (PM), and PM exposure with concurrent treadmill exercise (PME). Notably, the PM group exhibited markedly lower alpha diversity and richness compared to the Con group and our analysis of beta diversity revealed significant variations among the intervention groups. Members of the Lachnospiraceae family showed significant enhancement in the exercise intervention groups (EXE and PME) compared to the Con and PM groups. The biomarker Lactobacillus, Coriobacteraceae, and Anaerofustis were enriched in the EXE group, while Desulfovibrionaceae, Mucispirillum schaedleri, Lactococcus and Anaeroplasma were highly enriched in the PM group. Differential abundance analysis revealed that Paraprevotella, Bacteroides, and Blautia were less abundant in the 12-week PM exposure group than in the 3-day PM exposure group. Moreover, both the 3-day and 12-week PM exposure groups exhibited a reduced relative abundance of Bacteroides uniformis, SMB53, and Staphylococcus compared to non-PM exposure groups. These findings will help delineate the possible roles and associations of altered microbiota resulting from the studied interventions, paving the way for future mechanistic research.

Taxifolin as a Metallo-ß-Lactamase Inhibitor in Combination with Augmentin against Verona Imipenemase 2 Expressing Pseudomonas aeruginosa.

Among the various mechanisms that bacteria use to develop antibiotic resistance, the multiple expression of ß-lactamases is particularly problematic, threatening public health and increasing patient mortality rates. Even if a combination therapy-in which a ß-lactamase inhibitor is administered together with a ß-lactam antibiotic-has proven effective against serine-ß-lactamases, there are no currently approved metallo-ß-lactamase inhibitors. Herein, we demonstrate that quercetin and its analogs are promising starting points for the further development of safe and effective metallo-ß-lactamase inhibitors. Through a combined computational and in vitro approach, taxifolin was found to inhibit VIM-2 expressing P. aeruginosa cell proliferation at <4 µg/mL as part of a triple combination with amoxicillin and clavulanate. Furthermore, we tested this combination in mice with abrasive skin infections. Together, these results demonstrate that flavonol compounds, such as taxifolin, may be developed into effective metallo-ß-lactamase inhibitors.

A novel strategy to characterize the pattern of ß-lactam antibiotic-induced drug resistance in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an urgent public health threat, according to the CDC. This pathogen has few treatment options and causes severe nosocomial infections with > 50% fatality rate. Although previous studies have examined the proteome of CRAb, there have been no focused analyses of dynamic changes to ß-lactamase expression that may occur due to drug exposure. Here, we present our initial proteomic study of variation in ß-lactamase expression that occurs in CRAb with different ß-lactam antibiotics. Briefly, drug resistance to Ab (ATCC 19606) was induced by the administration of various classes of ß-lactam antibiotics, and the cell-free supernatant was isolated, concentrated, separated by SDS-PAGE, digested with trypsin, and identified by label-free LC-MS-based quantitative proteomics. Thirteen proteins were identified and evaluated using a 1789 sequence database of Ab ß-lactamases from UniProt, the majority of which were Class C ß-lactamases (≥ 80%). Importantly, different antibiotics, even those of the same class (e.g. penicillin and amoxicillin), induced non-equivalent responses comprising various isoforms of Class C and D serine-ß-lactamases, resulting in unique resistomes. These results open the door to a new approach of analyzing and studying the problem of multi-drug resistance in bacteria that rely strongly on ß-lactamase expression.

Cryo-EM structure of amyloid fibril formed by α-synuclein hereditary A53E mutation reveals a distinct protofilament interface.

Synucleinopathies like Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA), have the same pathologic feature of misfolded α-synuclein protein (α-syn) accumulation in the brain. PD patients who carry α-syn hereditary mutations tend to have earlier onset and more severe clinical symptoms than sporadic PD patients. Therefore, revealing the effect of hereditary mutations to the α-syn fibril structure can help us understand these synucleinopathies' structural basis. Here, we present a 3.38 Å cryo-electron microscopy structure of α-synuclein fibrils containing the hereditary A53E mutation. The A53E fibril is symmetrically composed of two protofilaments, similar to other fibril structures of WT and mutant α-synuclein. The new structure is distinct from all other synuclein fibrils, not only at the interface between proto-filaments, but also between residues packed within the same proto-filament. A53E has the smallest interface with the least buried surface area among all α-syn fibrils, consisting of only two contacting residues. Within the same protofilament, A53E reveals distinct residue re-arrangement and structural variation at a cavity near its fibril core. Moreover, the A53E fibrils exhibit slower fibril formation and lower stability compared to WT and other mutants like A53T and H50Q, while also demonstrate strong cellular seeding in α-synuclein biosensor cells and primary neurons. In summary, our study aims to highlight structural differences - both within and between the protofilaments of A53E fibrils - and interpret fibril formation and cellular seeding of α-synuclein pathology in disease, which could further our understanding of the structure-activity relationship of α-synuclein mutants.

A novel strategy to characterize the pattern of ß-lactam antibiotic-induced drug resistance in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an urgent public health threat, according to the CDC. This pathogen has few treatment options and causes severe nosocomial infections with > 50% fatality rate. Although previous studies have examined the proteome of CRAb, there have been no focused analyses of dynamic changes to ß-lactamase expression that may occur due to drug exposure. Here, we present our initial proteomic study of variation in ß-lactamase expression that occurs in CRAb with different ß-lactam antibiotics. Briefly, drug resistance to Ab (ATCC 19606) was induced by the administration of various classes of ß-lactam antibiotics, and the cell-free supernatant was isolated, concentrated, separated by SDS-PAGE, digested with trypsin, and identified by label-free LC-MS-based quantitative proteomics. Peptides were identified and evaluated using a 1789 sequence database of Ab ß-lactamases from UniProt. Importantly, we observed that different antibiotics, even those of the same class ( e.g. penicillin and amoxicillin), induce non-equivalent responses comprising various Class C and D serine-ß-lactamases, resulting in unique resistomes. These results open the door to a new approach of analyzing and studying the problem of multi-drug resistance in bacteria that rely strongly on ß-lactamase expression.