ACS Infectious Diseases最新文献

Antimicrobial resistance threatens health, and new agents are needed. Lefamulin is the only approved antibiotic of a new class in two decades. It targets the 50S peptidyl transferase center (PTC). Its efficacy against multidrug-resistant pathogens is limited by anionic envelopes, limiting penetration. We report a charge-anchored pleuromutilin exploiting an electrostatic Trojan horse to breach barriers and engage the ribosome. An N-pyridinium provides a cationic localizer. A two-step mechanism operates. Long-range electrostatics enrich ligands at anionic interfaces and the PTC. Short-range interactions secure high-affinity placement of the tricyclic core. Computer simulations support the occupancy of the activity pocket and the field-guided orientation of the cationic side chain for PNY-6b. Proteomics highlights ribosomal proteins as dominant targets. Cellular assays show biofilm eradication and membrane depolarization. In murine infections, PNY-6b lowers burden and improves survival. Electrostatic complementarity offers a generalizable design principle for targets with electrostatic fields and for pathogens with poor envelope permeability.

The COVID-19 pandemic has exposed the limitations of traditional vaccine development models: these approaches rely excessively on pathogen-specific antigen design, feature lengthy development cycles, and struggle to address threats from rapidly mutating pathogens and emerging pathogens. Even before the pandemic, certain traditional vaccines (such as BCG) demonstrated "cross-protection" effects beyond their target diseases. The trained immunity (TRIM) theory offers a promising path to develop broad-spectrum, effective, and durable vaccines. This review summarizes core advances in TRIM within vaccinology, systematically outlining vaccine design strategies based on this concept for the first time. These strategies encompass vaccine-mediated cross-protection, methods to enhance vaccine potency and persistence, pathways to achieve broad-spectrum effects, and regulatory characteristics involving immune recognition, antigen delivery, safety, and tolerability. This study explores the synergistic effects and application prospects of TRIM adjuvants such as β-glucan and Toll-like receptor (TLR) agonists. The impact of transgenerational immune effects on offspring immune function provides a crucial direction for future research. It also highlights current limitations in studies regarding persistence, individual variability, and risks of excessive inflammation. Existing vaccines capable of inducing TRIM will inspire next-generation vaccine development. Innovative applications of this vaccine category can propel the advancement of trained immunity-based vaccines (TIbVs). This review proposes an innovative approach─the "Vaccine Immunity Foundation Hypothesis." This lays the groundwork for designing next-generation vaccines and advancing the clinical translation of TRIM therapies, establishing a theoretical foundation for developing broad-spectrum, highly effective, durable, and safe immune protection strategies.

Neutrophil extracellular traps (NETs) are crucial innate immune components that ensnare and neutralize pathogens. Inspired by this, we engineered a novel peptide, RFC, designed to mimic NETs' "trap-and-kill" strategy against Staphylococcus aureus infections. RFC integrates an antimicrobial peptide (KR12), a self-assembling motif (KLVFF), and a Staphylococcus-targeting sequence (CARGGLKSC). In vitro, RFC exhibited potent broad-spectrum activity (minimum inhibitory concentration (MIC) as low as 4 μM), fast bactericidal kinetics (>3-log₁₀ reduction within 2 h at 1× MIC), inhibited biofilm formation (>92% at 2× MIC), and eradicated persister cells, while showing high biocompatibility. RFC self-assembles into nanofibrillar networks for bacterial entrapment and disrupts membranes. In vivo, RFC potently treated murine polymicrobial skin infections (99.3% wound closure) and lethal sepsis, improving survival from 16.6% to 66.7%, clearing bacteremia, and suppressing cytokines without toxicity. These findings highlight RFC as a promising antimicrobial agent, combining bacterial targeting, killing, and aggregation with tissue healing and immune activation capabilities, offering a novel strategy against challenging S. aureus infections.

We investigated the activity of a series of 75 lipophilic bisphosphonates against Candida glabrata. Thirty-six compounds had MIC < 1 μg/mL, 18 had MIC < 0.5 μg/mL, and 2 had MIC = 0.13 μg/mL, comparable to amphotericin B and caspofungin. The lipophilic bisphosphonates were ∼20-fold more potent against C. glabrata than the most potent hydrophilic bisphosphonate, zoledronate. The most active compounds were pyridinium bisphosphonates followed by imidazolium bisphosphonates, while aryl bisphosphonates were less active. Several compounds had selectivity index values against a human cell line in the 1000-3600 range, the most selective compounds being para-substituted pyridinium bisphosphonates. We also found similar activity against a caspofungin-resistant FKS2 (F659S) mutant. Some combinations of lipophilic bisphosphonates had synergistic activity with FICI values in the ∼0.3-0.5 range, and similar synergies were observed with fluconazole, implicating ergosterol biosynthesis inhibition leading to compromised membrane structure and function. Cell growth inhibition was rescued by ascorbic acid, glutathione, and N-acetyl cysteine, indicating a ROS-based killing mechanism. There was also synergy with other antifungals but very strong antagonism with verapamil (FICI ∼4), which blocks calcium channels. Unlike hydrophilic bisphosphonates, which target farnesyl diphosphate synthase, lipophilic bisphosphonates also target squalene synthase, suggesting that the combination of multitargeting bisphosphonates is one origin of the synergistic interactions observed. Given that one of the lipophilic bisphosphonates studied here (BPH-1237) has been shown to have activity against many other human fungal pathogens, combinations with the compounds described here may be of interest as antifungal leads.

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.