ACS Infectious Diseases最新文献

Gram-negative bacterial infections are characterized by the release of lipopolysaccharide (LPS), a key outer membrane component that triggers a robust host immune response via TLR4 signaling. In this study, three series of dual-target (LpxC/PD-L1) inhibitors were rationally designed via a structural splicing approach, synthesized, and evaluated for their in vitro biological activities. Among them, compound 12d displayed potent antibacterial activity and significant dual-target (LpxC/PD-L1) inhibitory efficacy. To improve its bioavailability and targeting capability, the nanocomposite (NC-12d) was further constructed to sense the infection microenvironment. Subsequent in vivo evaluation confirmed the dual therapeutic functions of these agents: effective bacterial suppression and immune activation, which collectively accelerated host recovery from drug-resistant bacterial infection. In summary, this study not only broadens the scope of antibacterial drug development but also offers a drug delivery pathway for the treatment of bacterial infections.

Interest in antimicrobial peptides has increased dramatically over the last few decades as researchers continue to explore their potential as alternatives to small molecules, as well as their applications in agriculture and food preservation. One promising yet small antimicrobial peptide class is that consisting of a single β-hairpin cyclized via intramolecular disulfide bonds, commonly termed β-hairpin antimicrobial peptides (β-AMPs). Their short length constrained cyclic structure and wide range of activities make them exciting to the general scientific community and drug developers alike; however, despite being found across several phyla, there remain fewer than 30 identified sequence families, making them exceedingly rare relative to more common structural classes. In this review, we identify and describe 27 unique macrocyclic β-AMP sequence families from the literature, with an emphasis on newer and lesser-known families. We then analyze the class’s sequence composition both as a whole and broken down by structural region, finding common characteristics including lengths of 11–25 amino acids, cationic charge, two or more cysteine pairs separated by at least three residues, and strong enrichment for arginine relative to lysine. We then discuss strategies for using these sequence characteristics to help expand the class and improve their relative underrepresentation.

The 23S rRNA methylating enzyme Cfr, found in pathogens including Staphylococcus aureus, Clostridium difficile, Escherichia coli, and Klebsiella pneumoniae, confers resistance to phenicols, lincosamides, oxazolidinones (including linezolid), pleuromutilins, and streptogramins A (the PhLOPS_A phenotype). Cfr catalyzes methylation of the C8 position of the A2503 base in 23S rRNA, the recognition site of the above antibiotic classes. Along with the RlmN housekeeping enzyme, Cfr can also promote methylation of the C2 position of the same base. The molecular and structural basis of Cfr’s dual substrate specificity is not known, which hinders our ability to design Cfr-targeting inhibitors necessary to curb PhLOPS_A resistance. Here, we present the first crystal structure of Cfr and a detailed analysis of its possible interactions with rRNA. Using structure-guided mutagenesis, mass spectrometry analysis of in cellulo 23S rRNA methylated species, and in cellulo resistance studies, we identify the key amino acids essential for Cfr methylation and multidrug resistance activity. In particular, we found that Cfr’s Q329 residue is important for C8-specific methylation. These data provide a framework for further studies of the biochemistry, structure, and inhibition of this important resistance determinant.

Enzymes that depend on the cofactor pyridoxal 5′-phosphate (PLP) catalyze a remarkable variety of biochemical reactions in all organisms. In particular, the genome of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), encodes 45 bona fide PLP-dependent enzymes plus a few related proteins that presumably do not have enzymic function. The large majority of the 45 enzymes have been characterized in terms of catalytic activity and structure. Several of them have been shown to be central to the bacterium’s survival and pathogenicity, while some of these enzymes are targets of an extant drug (d-cycloserine). Herein, the annotated catalog of the PLP-dependent enzymes in M. tuberculosis is presented and analyzed with three main goals in mind. The first will be to assess the specific aspects of mycobacterial metabolism that rely most on PLP-dependent enzymes. A second goal will be to signal those enzymes whose function is still uncertain and whose functional characterization may help to further understand the biology of M. tuberculosis. Finally, we will examine the potential and limitations of targeting the PLP-dependent enzymes for the development of new antimycobacterial drugs.

Hepatitis E virus (HEV) causes significant global disease burden with no approved targeted therapies, highlighting the urgent need for innovative treatment strategies. G-quadruplexes (G4s), noncanonical nucleic acid structures formed by guanine-rich sequences, have emerged as important regulators of viral replication. Here, we identified two potential G4 sequences within HEV negative-sense genomic RNA. Circular dichroism spectroscopy confirmed their stable, parallel G4 structures, with structural stability enhanced by the G4-binding ligand pyridostatin (PDS). Using an EGFP reporter system, we demonstrated that these G4s significantly suppressed downstream gene expression, an effect potentiated by PDS treatment. In HEV infection models, PDS substantially inhibited viral RNA synthesis and ORF2 protein expression. This antiviral activity was recapitulated by the structurally distinct G4-binding ligand TMPyP4, but not by the weak-binding control TMPyP2, confirming G4-dependent regulation. Our findings establish G4s as functional regulatory elements in the HEV life cycle and as promising RNA-targeted therapeutic targets against HEV.

Efflux pumps operating in bacteria continuously evolve and contribute significantly toward the rising global trends in antimicrobial resistance (AMR). Our earlier studies demonstrated that the expression of tripartite resistance nodulation division (RND) efflux pump containing the outer membrane protein (OMP), membrane fusion protein (MFP), and inner RND pump from different Gram-negative bacteria results in elevated minimum inhibitory concentrations (MICs) of different antibiotics. Interestingly, parts of this complex could be transferred either within the species or across genera. Despite limited sequence homology, we report the existence of significant structural and functional conservation between the distantly related MFP and RND proteins. Following the assembly of MFP components (AcrA, MexA, OqxA) and RND components (AcrB, MexB, OqxB) from E. coli, P. aeruginosa, and K. pneumoniae, respectively, we report evidence of functioning efflux pumps using real-time Nile Red assays and enhanced biofilm formation. Further substantiation of the latter is provided through docking and molecular dynamics (MD) simulation studies, which offer insights about the direct interactions of RND efflux pumps with AI-2, the major quorum-sensing molecule of E. coli. Results described here implicate that after transmission, possibly via horizontal gene transfer or e-DNA within bacteria, the assembled efflux pump components could drive multiple aspects of AMR, including its dissemination and ability to adapt to alternate lifestyles such as biofilms, facilitating better survival.

Gyrase and topoisomerase IV, enzymes that play critical roles during DNA replication, are the targets of fluoroquinolones and other antibacterials. Gyrase removes positive supercoils that accumulate ahead of replication forks, while topoisomerase IV untangles daughter chromosomes. Although topoisomerase IV is an essential enzyme in most bacteria, some species, including Mycobacterium tuberculosis and Mycobacteroides abscessus, encode only gyrase. In these species, gyrase is the sole target of fluoroquinolones and is believed to assume the cellular functions of both type II topoisomerases. Although fluoroquinolones and emerging antibacterials such as spiropyrimidinetriones induce gyrase-mediated DNA cleavage, there is evidence that inhibition of gyrase function also plays a role in drug-induced cell death under some circumstances. Therefore, we examined the effects of moxifloxacin and ciprofloxacin (fluoroquinolones) and zoliflodacin (spiropyrimidinetrione) on the three catalytic activities presumably carried out by gyrase in mycobacteria: decatenation of tangled DNA, negative supercoiling of relaxed DNA, and relaxation of positive supercoils. Under all circumstances, lower concentrations of antibacterials were required to inhibit intermolecular DNA decatenation as compared to the intramolecular DNA relaxation or supercoiling functions of gyrase. Differences in drug potency could not be attributed solely to rates of individual reactions or the DNA substrates utilized. Rather, results suggest that the potency of gyrase inhibition by interfacial antibacterials is modulated by the topological state of the DNA and its specific interactions with gyrase. Whereas most studies focus on DNA cleavage induced by gyrase-targeted antibacterials, this study provides mechanistic insights into how antibacterials rob replicating cells of essential gyrase functions.

Innate immune cells, such as monocytes and macrophages, provide the earliest defense against intracellular pathogen infection by initiating signaling pathways and restricting pathogen replication. However, the full complement of proteins that mediate cell-autonomous immunity remains incompletely defined. Here, we applied cysteine-directed activity-based protein profiling (ABPP) to map proteome-wide cysteine reactivity changes in THP-1 monocytes and primary human monocyte-derived macrophages during Mycobacterium tuberculosis (Mtb) infection. Across both cell types, we quantified 148 cysteine residues with altered reactivity. Knockdown of a subset of proteins harboring infection-induced reactivity significantly altered Mtb replication in THP-1 monocytes, linking proteins with reactive cysteines to antimicrobial defense. These data define previously unrecognized host protein changes during Mtb infection and provide a resource for investigating post-translational events that regulate innate immune responses to intracellular bacteria.

The development of innovative therapeutics against WHO priority pathogens is an urgent global need. Here, we demonstrate the functionalities of a human antibody, previously isolated by whole cell biopanning of the phage display scFv library. The yPgi3G4 IgG1 could mediate antibody-dependent phagocytosis of Pseudomonas aeruginosa by human matured THP-1 monocytes, of which colocalization with caveolin was clearly observed at 24 h. Next, the antibacterial activity against live P. aeruginosa was demonstrated by an agglutination assay and complement-mediated killing of the bacteria. Lipopolysaccharide (LPS) extraction and Western blot (WB) analysis suggested that LPS was the target of the yPgi3G4 IgG1 antibody. A cross-reactivity assay to available isolates in Thailand and France showed that the antibody could detect 6 P. aeruginosa serotypes including several multidrug-resistant clinical isolates. This study proved the potential of using this strategy to identify a biotherapeutic for a certain quotient of multidrug-resistant P. aeruginosa and other bacterial infections.

Malaria remains a major global health threat, and the emergence of partial artemisinin resistance challenges current treatment regimens. Reliable antimalarial screening assays are therefore essential for identifying new drug candidates. The parasite reduction ratio (PRR) assay provides valuable pharmacodynamic insights but is limited by its labor-intensive, 14- to 28-day incubation period. We developed an optimized PRR assay protocol using the highly sensitive chemiluminescence-based lacZ/β-gal^SENSOR readout, reducing assay incubation duration to 7 days while maintaining informative pharmacodynamic parameters, including lag phase, parasite clearance time, parasite reduction ratio, and maximum killing effect. In contrast, the [³H]-hypoxanthine incorporation method failed to detect viable parasites reliably and consistently overestimated drug activity with the shortened protocol. This novel lacZ/β-gal^SENSOR PRR assay enables laboratories without access to radioactive facilities to evaluate antimalarial compounds efficiently, providing robust time–killing profiles with greater convenience, higher throughput, and lower equipment requirements than existing readout methods.