Structure-Activity Relationship for the Oxadiazole Class of Antibiotics

The structure-activity relationship (SAR) for the newly discovered oxadiazole class of antibiotics is described with evaluation of 120 derivatives of the lead structure. This class of antibiotics was discovered by in silico docking and scoring against the crystal structure of a penicillin-binding protein. They impair cell-wall biosynthesis and exhibit activities against the Gram-positive bacterium Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant and linezolid-resistant S. aureus. 5-(1H-Indol-5-yl)-3-(4-(4-(trifluoromethyl)phenoxy)phenyl)-1,2,4-oxadiazole (antibiotic 75b) was efficacious in a mouse model of MRSA infection, exhibiting a long half-life, a high volume of distribution, and low clearance. This antibiotic is bactericidal and is orally bioavailable in mice. This class of antibiotics holds great promise in recourse against infections by MRSA.Fil: Spink, Edward. University of Notre Dame-Indiana; Estados UnidosFil: Ding, Derong. University of Notre Dame-Indiana; Estados UnidosFil: Peng, Zhihong. University of Notre Dame-Indiana; Estados UnidosFil: Boudreau, Marc A.. University of Notre Dame-Indiana; Estados UnidosFil: Leemans, Erika. University of Notre Dame-Indiana; Estados UnidosFil: Lastochkin, Elena. University of Notre Dame-Indiana; Estados UnidosFil: Song, Wei. University of Notre Dame-Indiana; Estados UnidosFil: Lichtenwalter, Katerina. University of Notre Dame-Indiana; Estados UnidosFil: O’Daniel, Peter I.. University of Notre Dame-Indiana; Estados UnidosFil: Testero, Sebastian Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina. University of Notre Dame-Indiana; Estados UnidosFil: Pi, Hualiang. University of Notre Dame-Indiana; Estados UnidosFil: Schroeder, Valerie A.. University of Notre Dame-Indiana; Estados UnidosFil: Wolter, William R.. University of Notre Dame-Indiana; Estados UnidosFil: Antunes, Nuno T.. University of Notre Dame-Indiana; Estados UnidosFil: Suckow, Mark A.. University of Notre Dame-Indiana; Estados UnidosFil: Vakulenko, Sergei. University of Notre Dame-Indiana; Estados UnidosFil: Chang, Mayland. University of Notre Dame-Indiana; Estados UnidosFil: Mobashery, Shahriar. University of Notre Dame-Indiana; Estados Unido

A Putative P-Type ATPase Required for Virulence and Resistance to Haem Toxicity in Listeria monocytogenes

Regulation of iron homeostasis in many pathogens is principally mediated by the ferric uptake regulator, Fur. Since acquisition of iron from the host is essential for the intracellular pathogen Listeria monocytogenes, we predicted the existence of Fur-regulated systems that support infection. We examined the contribution of nine Fur-regulated loci to the pathogenicity of L. monocytogenes in a murine model of infection. While mutating the majority of the genes failed to affect virulence, three mutants exhibited a significantly compromised virulence potential. Most striking was the role of the membrane protein we designate FrvA (Fur regulated virulence factor A; encoded by frvA [lmo0641]), which is absolutely required for the systemic phase of infection in mice and also for virulence in an alternative infection model, the Wax Moth Galleria mellonella. Further analysis of the ΔfrvA mutant revealed poor growth in iron deficient media and inhibition of growth by micromolar concentrations of haem or haemoglobin, a phenotype which may contribute to the attenuated growth of this mutant during infection. Uptake studies indicated that the ΔfrvA mutant is unaffected in the uptake of ferric citrate but demonstrates a significant increase in uptake of haem and haemin. The data suggest a potential role for FrvA as a haem exporter that functions, at least in part, to protect the cell against the potential toxicity of free haem

IRON HOMEOSTATIC SYSTEMS AND IRON LIMITATION RESPONSES IN BACTERIA

Iron is required for most bacteria but its bioavailability is extremely low. So iron acquisition has become a major challenge in bacterial physiology. Iron acquisition includes uptake systems for elemental iron, ferric citrate, and various ferric-siderophore complexes. Iron-mediated regulation takes place at multi-levels: transcriptional, post-transcriptional, and translational. These regulatory systems enable bacteria to achieve homeostatic balance with iron (Chapter 1). Iron is also toxic at elevated levels. Recent results revealed that Fe(II) exporters play a crucial role in preventing iron overload. These include P1B-type ATPases, cation diffusion facilitators, major facilitator superfamily proteins, and membrane bound ferritin-like proteins (Chapter 2). Among these systems, FrvA is a virulence factor in Listeria monocytogenes. The characterization of FrvA as an Fe(II) efflux transporter provides the first direct evidence linking iron efflux to bacterial pathogenesis. Furthermore, FrvA is a high-affinity Fe(II) exporter and its expression imposes severe iron starvation in Bacillus subtilis (Chapter 3). Thus it has been employed as an inducible genetic tool to study iron limitation responses. Iron acquisition and homeostasis systems need to be tightly regulated to ensure sufficiency for biological functions but not excess that would trigger intoxication. The ferric uptake regulator (Fur) monitors intracellular iron levels and plays a central role in maintaining bacterial iron homeostasis. However, it is unclear whether Fur-regulated genes are derepressed coordinately or in a sequential manner upon iron starvation. Here the iron limitation responses were characterized in B. subtilis (Chapter 4). In particular, the Fur-regulated genes are induced in three sequential waves in response to iron depletion: (i) cells increase their capacity for iron import from common sources of iron in the environment; (ii) cells turn on high-affinity siderophore-mediated import systems to scavenge iron; (iii) as iron levels decrease further, cells activate an iron-sparing response to remodel their proteome. This graded response correlates with in vivo occupancy of Fur protein and can be explained, at least in part, as a direct effect of differences in operator binding affinity of Fur protein. These results provide insights into the distinct roles of Fur-target genes and contribute to our understanding of bacterial metalloregulatory systems

Ferrous iron efflux systems in bacteria

Iron efflux transporters play a critical role in protecting cells from iron intoxication.</p

Genome-Wide Characterization of the Fur Regulatory Network Reveals a Link between Catechol Degradation and Bacillibactin Metabolism in Bacillus subtilis

Many bacteria synthesize high-affinity iron chelators (siderophores). Siderophore-mediated iron acquisition is an efficient and widely utilized strategy for bacteria to meet their cellular iron requirements. One prominent class of siderophores uses catecholate groups to chelate iron. B. subtilis bacillibactin, structurally similar to enterobactin (made by enteric bacteria), is a triscatecholate siderophore that is hydrolyzed to monomeric units after import to release iron. However, the ultimate fates of these catechol compounds and their potential toxicities have not been defined previously. We performed genome-wide identification of Fur binding sites in vivo and uncovered a connection between catechol degradation and bacillibactin metabolism in B. subtilis. Besides its role in the detoxification of environmental catechols, the catechol 2,3-dioxygenase encoded by catDE also protects cells from intoxication by endogenous bacillibactin-derived catechol metabolites under iron-limited conditions. These findings shed light on the degradation pathway and precursor recycling of the catecholate siderophores.The ferric uptake regulator (Fur) is the global iron biosensor in many bacteria. Fur functions as an iron-dependent transcriptional repressor for most of its regulated genes. There are a few examples where holo-Fur activates transcription, either directly or indirectly. Recent studies suggest that apo-Fur might also act as a positive regulator and that, besides iron metabolism, the Fur regulon might encompass other biological processes such as DNA synthesis, energy metabolism, and biofilm formation. Here, we obtained a genomic view of the Fur regulatory network in Bacillus subtilis using chromatin immunoprecipitation sequencing (ChIP-seq). Besides the known Fur target sites, 70 putative DNA binding sites were identified, and the vast majority had higher occupancy under iron-sufficient conditions. Among the new sites detected, a Fur binding site in the promoter region of the catDE operon is of particular interest. This operon, encoding catechol 2,3-dioxygenase, is critical for catechol degradation and is under negative regulation of CatR and YodB. These three repressors (Fur, CatR, and YodB) function cooperatively to regulate the transcription of catDE, with Fur functioning as a sensor of iron limitation and CatR as the major sensor of catechol stress. Genetic analysis suggests that CatDE is involved in metabolism of the catecholate siderophore bacillibactin, particularly when bacillibactin is constitutively produced and accumulates intracellularly, potentially generating endogenous toxic catechol derivatives. This study documents a role for catechol degradation in bacillibactin metabolism and provides evidence that catechol 2,3-dioxygenase can detoxify endogenously produced catechol substrates in addition to its more widely studied role in biodegradation of environmental aromatic compounds and pollutants

Dysregulation of Magnesium Transport Protects Bacillus subtilis against Manganese and Cobalt Intoxication

Cellular metal ion homeostasis is tightly regulated. When metal ion levels are imbalanced, or when one metal is at toxic levels, enzymes may bind to the wrong metal cofactor. Enzyme mismetallation can impair metabolism, lead to new and deleterious reactions, and cause cell death. Beginning with Bacillus subtilis strains genetically sensitized to metal intoxication through loss of efflux or by lowering intracellular iron, we identified mutations that suppress the deleterious effects of excess Mn(II) or Co(II). For both metals, mutations in mpfA , encoding a Mg(II) efflux pump, suppressed toxicity. These mutant strains have elevated intracellular Mg(II), suggesting that Mg(II)-dependent processes are very sensitive to disruption by transition metals. </jats:p

HiA: Towards Chinese Multimodal LLMs for Comparative High-Resolution Joint Diagnosis

Multimodal large language models (MLLMs) have been explored in the Chinese medical domain for comprehending complex healthcare. However, due to the flaws in training data and architecture design, current Chinese medical MLLMs suffer from several limitations: cultural biases from English machine translations, limited comparative ability from single image input and difficulty in identifying small lesions with low-resolution images. To address these problems, we first introduce a new instruction-following dataset, Chili-Joint (Chinese Interleaved Image-Text Dataset for Joint Diagnosis) collected from the hospital in mainland China, avoiding cultural biases and errors caused by machine translation. Besides one single image input, Chili-Joint also has multiple images obtained at various intervals during a patient’s treatment, thus facilitating an evaluation of the treatment’s outcomes. We further propose a novel HiA (High-resolution instruction-aware Adapter) to incorporate high-resolutioninstruction-aware visual features into LLMs to facilitate the current MLLMs to observe the small lesions as well as the comparative analysis. Extensive experiments on Chili-Joint demonstrate our HiA can be a plug-and-play method to improve the performance of current MLLMs for medical analysis. The code is available at https://github.com/xmed-lab/HiA

The extracytoplasmic sigma factor σX supports biofilm formation and increases biocontrol efficacy in Bacillus velezensis 118

Abstract Plant growth promoting rhizobacteria (PGPR) offer an environmentally friendly and sustainable approach to combat pathogens and enhance crop production. The biocontrol activity of PGPR depends on their ability to colonize plant roots and synthesize antimicrobial compounds that inhibit pathogens. However, the regulatory mechanisms underlying these processes remain unclear. In this study, we isolated and characterized Bacillus velezensis 118, a soil isolate that exhibits potent biocontrol activity against Fusarium wilt of banana. Deletion of sigX, encoding an extracytoplasmic function (ECF) sigma factor previously implicated in controlling biofilm architecture in B. subtilis, reduced biocontrol efficacy. The B. velezensis 118 sigX mutant displayed reduced biofilm formation but had only a minor defect in swarming motility and a negligible impact on lipopeptide production. These findings highlight the importance of regulatory processes important for root colonization in the effectiveness of Bacillus spp. as biocontrol agents against phytopathogens

A Statistical Thermodynamic Model for Ligands Interacting With Ion Channels: Theoretical Model and Experimental Validation of the KCNQ2 Channel

Ion channels are important therapeutic targets, and their pharmacology is becoming increasingly important. However, knowledge of the mechanism of interaction of the activators and ion channels is still limited due to the complexity of the mechanisms. A statistical thermodynamic model has been developed in this study to characterize the cooperative binding of activators to ion channels. By fitting experimental concentration-response data, the model gives eight parameters for revealing the mechanism of an activator potentiating an ion channel, i.e., the binding affinity (KA), the binding cooperative coefficients for two to four activator molecules interacting with one channel (γ, μ, and ν), and the channel conductance coefficients for four activator binding configurations of the channel (a, b, c, and d). Values for the model parameters and the mechanism underlying the interaction of ztz240, a proven KCNQ2 activator, with the wild-type channel have been obtained and revealed by fitting the concentration-response data of this activator potentiating the outward current amplitudes of KCNQ2. With these parameters, our model predicted an unexpected bi-sigmoid concentration-response curve of ztz240 activation of the WT-F137A mutant heteromeric channel that was in good agreement with the experimental data determined in parallel in this study, lending credence to the assumptions on which the model is based and to the model itself. Our model can provide a better fit to the measured data than the Hill equation and estimates the binding affinity, as well as the cooperative coefficients for the binding of activators and conductance coefficients for binding states, which validates its use in studying ligand-channel interaction mechanisms