Repurposing based identification of novel inhibitors against mmps5-mmpl5 efflux pump of Mycobacterium smegmatis: A combined in silico and in vitro study

In the current era of a pandemic, infections of COVID-19 and Tuberculosis (TB) enhance the detrimental effects of both diseases in suffering individuals. The resistance mechanisms evolving in Mycobacterium tuberculosis are limiting the efficiency of current therapeutic measures and pressurizing the stressed medical infrastructures. The bacterial efflux pumps enable the development of resistance against recently approved drugs such as bedaquiline and clofazimine. Consequently, the MmpS5-MmpL5 protein system was selected because of its role in efflux pumping of anti-TB drugs. The MmpS5-MmpL5 systems of Mycobacterium smegmatis were modelled and the virtual screening was performed using an ASINEX library of 5968 anti-bacterial compounds. The inhibitors with the highest binding affinities and QSAR based highest predicted inhibitory concentration were selected. The MmpS5-MmpL5 associated systems with BDE_26593610 and BDD_27860195 showed highest inhibitory parameters

Transcriptomic profile of mycobacterium smegmatis in response to an imidazo[1,2-b][1,2,4,5]tetrazine reveals its possible impact on iron metabolism

Tuberculosis (TB), caused by the Mycobacterium tuberculosis complex bacteria, is one of the most pressing health problems. The development of new drugs and new therapeutic regimens effective against the pathogen is one of the greatest challenges in the way of tuberculosis control. Imidazo[1,2-b][1,2,4,5]tetrazines have shown promising activity against M. tuberculosis and M. smegmatis strains. Mutations in MSMEG_1380 lead to mmpS5–mmpL5 operon overexpression, which provides M. smegmatis with efflux-mediated resistance to imidazo[1,2-b][1,2,4,5]tetrazines, but the exact mechanism of action of these compounds remains unknown. To assess the mode of action of imidazo[1,2-b][1,2,4,5]tetrazines, we analyzed the transcriptomic response of M. smegmatis to three different concentrations of 3a compound: 1/8×, 1/4×, and 1/2× MIC. Six groups of genes responsible for siderophore synthesis and transport were upregulated in a dose-dependent manner, while virtual docking revealed proteins involved in siderophore synthesis as possible targets for 3a

The use of omics technologies in creating LBP and postbiotics based on the Limosilactobacillus fermentum U-21

In recent years, there has been an increasing tendency to create drugs based on certain commensal bacteria of the human microbiota and their ingredients, primarily focusing on live biotherapeutics (LBPs) and postbiotics. The creation of such drugs, termed pharmacobiotics, necessitates an understanding of their mechanisms of action and the identification of pharmacologically active ingredients that determine their target properties. Typically, these are complexes of biologically active substances synthesized by specific strains, promoted as LBPs or postbiotics (including vesicles): proteins, enzymes, low molecular weight metabolites, small RNAs, etc. This study employs omics technologies, including genomics, proteomics, and metabolomics, to explore the potential of Limosilactobacillus fermentum U-21 for innovative LBP and postbiotic formulations targeting neuroinflammatory processes. Proteomic techniques identified and quantified proteins expressed by L. fermentum U-21, highlighting their functional attributes and potential applications. Key identified proteins include ATP-dependent Clp protease (ClpL), chaperone protein DnaK, protein GrpE, thioredoxin reductase, LysM peptidoglycan-binding domain-containing protein, and NlpC/P60 domain-containing protein, which have roles in disaggregase, antioxidant, and immunomodulatory activities. Metabolomic analysis provided insights into small-molecule metabolites produced during fermentation, revealing compounds with anti-neuroinflammatory activity. Significant metabolites produced by L. fermentum U-21 include GABA (γ-aminobutyric acid), niacin, aucubin, and scyllo-inositol. GABA was found to stabilize neuronal activity, potentially counteracting neurodegenerative processes. Niacin, essential for optimal nervous system function, was detected in vesicles and culture fluid, and it modulates cytokine production, maintaining immune homeostasis. Aucubin, an iridoid glycoside usually secreted by plants, was identified as having antioxidant properties, addressing issues of bioavailability for therapeutic use. Scyllo-inositol, identified in vesicles, acts as a chemical chaperone, reducing abnormal protein clumps linked to neurodegenerative diseases. These findings demonstrate the capability of L. fermentum U-21 to produce bioactive substances that could be harnessed in the development of pharmacobiotics for neurodegenerative diseases, contributing to their immunomodulatory, anti-neuroinflammatory, and neuromodulatory activities. Data of the HPLC-MS/MS analysis are available via ProteomeXchange with identifier PXD050857

Repurposing Based Identification of Novel Inhibitors against MmpS5-MmpL5 Efflux Pump of <i>Mycobacterium smegmatis</i>: A Combined In Silico and In Vitro Study

In the current era of a pandemic, infections of COVID-19 and Tuberculosis (TB) enhance the detrimental effects of both diseases in suffering individuals. The resistance mechanisms evolving in Mycobacterium tuberculosis are limiting the efficiency of current therapeutic measures and pressurizing the stressed medical infrastructures. The bacterial efflux pumps enable the development of resistance against recently approved drugs such as bedaquiline and clofazimine. Consequently, the MmpS5-MmpL5 protein system was selected because of its role in efflux pumping of anti-TB drugs. The MmpS5-MmpL5 systems of Mycobacterium smegmatis were modelled and the virtual screening was performed using an ASINEX library of 5968 anti-bacterial compounds. The inhibitors with the highest binding affinities and QSAR based highest predicted inhibitory concentration were selected. The MmpS5-MmpL5 associated systems with BDE_26593610 and BDD_27860195 showed highest inhibitory parameters. These were subjected to 100 ns Molecular Dynamics simulations and provided the validation regarding the interaction studies. The in vitro studies demonstrated that the BDE_26593610 and BDD_27860195 can be considered as active inhibitors for M. smegmatis MmpS5-MmpL5. The outcomes of this study can be utilized in other experimentation aimed at drug design and discovery against the drug resistance strains of M. tuberculosis

Identification of Mutations Conferring Tryptanthrin Resistance to Mycobacterium smegmatis

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a global burden, responsible for over 1 million deaths annually. The emergence and spread of drug-resistant M. tuberculosis strains (MDR-, XDR- and TDR-TB) is the main challenge in global TB-control, requiring the development of novel drugs acting on new biotargets, thus able to overcome the drug-resistance. Tryptanthrin is a natural alkaloid, with great therapeutic potential due to its simple way of synthesis and wide spectrum of biological activities including high bactericidal activity on both drug-susceptible and MDR M. tuberculosis strains. InhA was suggested as the target of tryptanthrins by in silico modeling, making it a promising alternative to isoniazid, able to overcome drug resistance provided by katG mutations. However, neither the mechanism of action of tryptanthrin nor the mechanism of resistance to tryptanthrins was ever confirmed in vitro. We show that the MmpS5-MmpL5 efflux system is able to provide resistance to tryptanthrins using an in-house test-system. Comparative genomic analysis of spontaneous tryptanthrin-resistant M. smegmatis mutants showed that mutations in MSMEG_1963 (EmbR transcriptional regulator) lead to a high-level resistance, while those in MSMEG_5597 (TetR transcriptional regulator) to a low-level one. Mutations in an MFS transporter gene (MSMEG_4427) were also observed, which might be involved in providing a basal level of tryptanthrins-resistance

Diet and the Gut Microbiome as Determinants Modulating Metabolic Outcomes in Young Obese Adults

Obesity, along with metabolic disorders such as dyslipidemia and insulin resistance, increases the risk of cardiovascular disease, diabetes, various cancers, and other non-communicable diseases, thereby contributing to higher mortality rates. The intestinal microbiome plays a crucial role in maintaining homeostasis and influencing human metabolism. This study enrolled 82 young obese individuals, who were stratified into groups with or without metabolic disturbances. No significant differences in the alpha or beta diversity of the microbiota were observed among the groups. Insulin resistance was characterized by an increase in the number of Adlercreutzia and Dialister as well as a decrease in Collinsella, Coprococcus and Clostridiales. The dyslipidemia and dyslipidemia+insulin resistance groups had no significant differences in the gut microbiota. Dietary patterns also influenced microbial composition, with high protein intake increasing Leuconostoc and Akkermansia, and high fiber intake boosting Lactobacillus and Streptococcus. The genus Erwinia was associated with increases in visceral fat and serum glucose as well as a decrease in high-density lipoprotein cholesterol. Our findings highlight a significant association between gut microbiota composition and metabolic disturbances in young obese individuals, and they suggest that dietary modifications may promote a healthy microbiome and reduce the risk of developing metabolic disorders

Synthesis and Characterization of Novel 2-Acyl-3-trifluoromethylquinoxaline 1,4-Dioxides as Potential Antimicrobial Agents

The emergence of drug resistance in pathogens leads to a loss of effectiveness of antimicrobials and complicates the treatment of bacterial infections. Quinoxaline 1,4-dioxides represent a prospective scaffold for search of new compounds with improved chemotherapeutic characteristics. Novel 2-acyl-3-trifluoromethylquinoxaline 1,4-dioxides with alteration of substituents at position 2 and 6 were synthesized via nucleophilic substitution with piperazine moiety and evaluated against a broad panel of bacteria and fungi by measuring their minimal inhibitory concentrations. Their mode of action was assessed by whole-genomic sequencing of spontaneous drug-resistant Mycobacterium smegmatis mutants, followed by comparative genomic analysis, and on an original pDualrep2 system. Most of the 2-acyl-3-trifluoromethylquinoxaline 1,4-dioxides showed high antibacterial properties against Gram-positive strains, including mycobacteria, and the introduction of a halogen atom in the position 6 of the quinoxaline ring further increased their activity, with 13c being the most active compound. The mode of action studies confirmed the DNA-damaging nature of the obtained quinoxaline 1,4-dioxides, while drug-resistance may be provided by mutations in redox homeostasis genes, encoding enzymes potentially involved in the activation of the compounds. This study extends views about the antimicrobial and antifungal activities of the quinoxaline 1,4-dioxides and can potentially lead to the discovery of new antibacterial drugs