Escherichia coli, a facultative anaerobic Gram-negative member of the Enterobacteriaceae, is an increasingly important opportunistic pathogen driven in part by rising resistance to clinically important antibiotics. Regulation of multidrug efflux systems by two-component signal transduction pathways, particularly the BaeSR system, plays a central role in this process. However, the functional residues governing signal transduction through the sensor kinase BaeS remain incompletely defined. In this study, we integrated domain prediction, homology-guided site-directed mutagenesis, in vitro protein purification, autophosphorylation assays, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR)-based transcriptional analysis of selected BaeSR-regulated genes to delineate key residues required for BaeS function. Sequence analysis identified His250 as a candidate autophosphorylation site and Asn364 as a conserved residue within the catalytic domain. Biochemical characterization of purified wild-type BaeS and an H250A mutant demonstrated that His250 is indispensable for autophosphorylation. Consistently, RT-qPCR analysis showed that BaeS activation markedly induced the transcription of BaeSR-regulated efflux-associated genes, whereas genetic deletion of baeS or selective disruption of kinase activity by the N364A mutation abolished this response. Together, these findings establish His250 as a key residue for BaeS autophosphorylation and identify Asn364 as essential for inducible BaeSR signaling and activation of resistance-associated target genes, thereby establishing an experimental framework for elucidating BaeSR-mediated efflux regulation and informing future studies of resistance regulatory networks and potential intervention strategies centered on key signaling nodes.

Human metapneumovirus (HMPV) is an important respiratory pathogen for which specific antiviral therapies are currently unavailable, and the mechanisms by which HMPV regulates host mitochondrial homeostasis remain poorly understood. Here, we investigated the role of HMPV nucleoprotein (N) in mitophagy regulation and mitochondrial quality control, with a focus on its association with selective autophagy-related factors. Using recombinant protein purification, surface plasmon resonance, co-immunoprecipitation, confocal microscopy, mitochondrial-targeted mitophagy reporter analysis, and immunoblotting, we examined the interaction between HMPV N and host autophagy-associated factors and evaluated its effects on mitophagy. We demonstrated that HMPV N interacts with TAX1BP1 and LC3, two key components of the selective autophagy machinery. HMPV N signals were spatially associated with mitochondrial regions, and HMPV N expression increased the spatial association of TAX1BP1-positive structures with mitochondria, while reducing the spatial association of LC3-positive structures with TOM20-positive mitochondria under stress conditions. Consistently, HMPV N expression reduced CCCP-induced mitochondrial delivery to acidic compartments and attenuated TOM20 degradation, while exerting limited effects on basal mitochondrial turnover. These findings identify HMPV N as a viral regulator of stress-induced mitophagy and suggest that its interactions with selective autophagy-associated factors contribute to altered mitochondrial homeostasis during infection.

Brucellosis, acting as a typical chronic zoonotic disease, is caused by the invasion of Brucella into the human body. Outer membrane protein 25 (Omp25), specifically localized on the Brucella membrane, is the main virulence factor of Brucella and participates in multiple links of the damage process. Omp25c, a porin protein of Brucella, is a paralog of Omp25 with high sequence identity. NADH dehydrogenase [ubiquinone] complex I assembly factor 2 (Ndufaf2) has a key function in cell energy metabolism, particularly in the formation and activity of the mitochondrial respiratory chain. Loss of Ndufaf2 results in oxidative stress and mitochondrial DNA (mtDNA) deletion. However, the functional relationship between Omp25c and Ndufaf2, the underlying mechanism of the proteins, remains unclear. In this work, we purified the Omp25c and Ndufaf2proteins. Our data revealed that Omp25c directly interacts with Ndufaf2, as determined using Biacore analysis. In addition, assays revealed that Ompa2c reshapes the host cell’s redox environment by decreasing the oxidized nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide (NAD⁺/NADH) ratioand adenosine triphosphate (ATP) production, whereas Ndufaf2 exerts an opposing regulatory effect; Co-expression results further revealed an antagonistic relationship between the two during metabolic processes. These findings provide a new perspective for elucidating the mechanisms of mitochondrial functional regulation in Brucella–host interactions and lay the theoretical and experimental foundation for drug development targeting metabolic interventions to eliminate intracellular pathogens.