ACS Infectious Diseases最新文献

Ribosomal RNA (rRNA) modification at a specific site is a prevalent resistance mechanism utilized by multidrug-resistant pathogens, leading to antimicrobial resistance (AMR). The erythromycin-resistant methyltransferase (Erm) methylates rRNA at the conserved A2058 position, imparting resistance to a broad class of antibiotics, including macrolides, lincosamides, and streptogramin B (MLS_B). However, inhibitors that are highly specific to Erm are scarce in the literature. Herein, we report high-affinity DNA aptamers discovered through in vitro selection that target pathogenic Erm42. The aptamers, Apt-E1 and Apt-E2, displayed nanomolar binding affinity for Erm42 and effectively inhibited the Erm42-mediated methylation of rRNA. Using DNase I footprinting assays, truncated versions of Apt-E1 and Apt-E2 were engineered. They exhibited comparable binding as well as enhanced specificity toward Erm42 when compared to other methyltransferases and DNA-binding proteins. This study provides a novel DNA aptamer-based strategy, paving the way for the development of aptamer-based therapeutic and diagnostic tools to combat AMR.

Negamycin is a natural product antibiotic discovered in 1970 and shown to have a Gram-negative spectrum of activity. It has served as the starting point in drug discovery efforts due in large part to its structural simplicity and novel mode of inhibition of the bacterial ribosome. It follows that negamycin does not show cross-resistance with other antibacterial agents that operate on the ribosome, whether this would be due to target modification, drug efflux, or drug metabolism. Because of the deficiencies of current drug regimens for the treatment of infections caused by Gram-negative pathogens, having a new agent brought to the infectious disease formulary represents a critical medical need, as has been promoted by the World Health Organization and other entities. Negamycin has been the subject of over 20 total syntheses, often highlighting stereoselective chemistry toward installing its two chiral centers on an acyclic chain. Novel synthetic methodologies thereby developed can stimulate the synthesis of novel analogs. With this, progress has been made in devising more potent analogs than negamycin. Structural work has determined that negamycin binds to the A-site of the 30S ribosome encounter complex with tRNA. Advancements have been made to understand the mechanism of transport of negamycin to the bacterial cytoplasm to enable engagement of the ribosome. This review surveys much of what has been published around negamycin and its analogs, including aspects of the biological spectrum of activity and mode of action as well as limitations that have held back clinical development.

Candida auris poses a significant healthcare challenge, particularly within immunosuppressed patients. This pathogen can colonize the skin and develop biofilms associated with increased antifungal drug resistance that are difficult to treat with a limited antifungal repertoire. Some adjuvant treatments have been investigated, such as photodynamic therapy (PDT), which employs a photosensitizer (PS) irradiated by light. However, most PSs available suffer from poor biofilm penetration. In this in vitro study, a nanocarrier system was proposed as a possible strategy to facilitate the methylene blue (MB) photosensitizer penetration into biofilm and improve PDT action against C. auris. For this, positively (MB-P) and negatively (MB-N) charged liposomes encapsulating MB were successfully fabricated. In the PDT results, both liposome formulations eradicated planktonic cells of C. auris at minimum fungicidal concentrations (MFC) equivalent to those of free MB. MB-loaded liposomes showed enhanced penetration within biofilms and reduced C. auris biofilm burden ∼2× more compared to free MB. Additionally, biofilm biomass was reduced up to 37% with MB-loaded liposomes, while free MB only achieved ∼3% reduction. Furthermore, PDT mediated by MB-P or MB-N led to the production of reactive oxygen species (ROS) 2× higher than free MB, leading to greater oxidative damage toward C. auris biofilms. Finally, the biocompatibility of MB-loaded liposomes was examined against mammalian fibroblasts; MB-loaded liposomes maintained ∼80% cell viability compared to ∼58% viability for free MB. Promisingly, MB-P and MB-N liposomes were able to enhance the in vitro activity of PDT on C. auris biofilms, inciting the development of in vivo studies to validate their efficacy and safety.

The protein kinase G (PknG) protein of Mycobacterium tuberculosis is known to disrupt phagosome-lysosome (P-L) fusion, enabling the bacteria to persist within the host. In our previous study, we demonstrated that PknG inhibits GTPase activity of Rab7l1 by interacting with its inactive form (Rab7l1-GDP) to block the Rab7l1-GDP/GTP transition. As a result, the active Rab7l1 protein fails to localize onto the phagosome, which prevents recruitment of downstream P-L fusion markers like Rab7l1, EEA1, LAMP1, and LAMP2 to the phagosome, leading to inhibition of P-L fusion. In this study, we show that Rho GDP dissociation inhibitor-1 (RGDI-1) is a GDP dissociation inhibitor (GDI) for Rab7l1. RGDI-1 associates with Rab7l1 and forms a stable complex in the presence of PknG. Rab7l1 serves as a scaffold that brings together both PknG and RGDI-1, allowing PknG to interact and phosphorylate RGDI-1. Kinasing of RGDI-1 prevents its dissociation from Rab7l1, resulting in decreased activity of Rab7l1 GTPase in PMA-induced THP-1 cells. Thus, PknG prevents release of RGDI-1 from Rab7l1, leading to reduced Rab7l1 GTPase activity. When RGDI-1 is absent, PknG fails to inhibit P-L fusion, resulting in decreased mycobacterial survival inside the PMA-induced THP-1 cells. Our data suggest that PknG targets RGDI-1 to inhibit Rab7l1-mediated P-L fusion and thereby promotes mycobacterial survival inside the PMA-induced THP-1 cells.

Intrabacterial drug accumulation, mediated by the bacterial permeability barrier, efflux, and intrabacterial drug metabolism, is of general significance to the interaction between small molecules and bacteria. For example, the ability of a small molecule to accumulate within a bacterium influences its ability to serve as a chemical probe of an intracellular protein target and/or its efficacy as an antibacterial drug discovery entity. A general method to quantitatively interrogate both intrabacterial drug accumulation and metabolism (IBDM) is presented for Gram-negative bacteria and exemplified with Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa in both single-compound and high-throughput formats. The liquid chromatography–mass spectrometry-based platform does not depend on drug labeling, and its utility is highlighted through the exploration of the relationship between drug accumulation and drug minimum inhibitory concentration (MIC) for both wild-type and efflux-deficient strains of E. coli and K. pneumoniae clinical and laboratory strains of varying degrees of drug resistance. Furthermore, an investigation of drug synergy implicates the selective enhancement of the accumulation of one drug by its partner therapy. Finally, a high-throughput format is validated and deployed, which provides a readily adaptable approach to screening assays. We anticipate the further applications of this platform to both the translational and the fundamental studies of the interactions of small molecules with bacteria.

Zoonotic spillover of sarbecoviruses to humans resulted in the SARS-CoV-1 outbreak in 2003 and the current COVID-19 pandemic caused by SARS-CoV-2. In both cases, the viral spike protein (S) is the principal target of neutralizing antibodies that prevent infection. Within the spike, the immunodominant receptor-binding domain (RBD) is the primary target of neutralizing antibodies in COVID-19 convalescent sera and vaccine recipients. We have constructed stabilized RBD derivatives of different sarbecoviruses: SARS-CoV-1 (Clade 1a), WIV-1 (Clade 1a), RaTG13 (Clade 1b), RmYN02 (Clade 2), and BtKY72 (Clade 3). Stabilization enhanced yield by 3–23-fold. The RBD derivatives were conformationally intact, as assayed by binding to multiple broadly neutralizing antibodies. The stabilized RBDs show significant enhancement in apparent T_m, exhibit resistance to a 2-h incubation at temperatures up to 60 °C in PBS in contrast to the corresponding WT RBDs, and show prolonged stability of over 15 days at 37 °C after lyophilization. In mice immunizations, both stabilization and trimerization significantly enhanced elicited neutralization titers by ∼100-fold. The stabilized RBD cocktail elicited highly neutralizing titers against both homologous and heterologous pseudoviruses. The immunogenicity of the vaccine formulation was assessed in both naïve and SARS-CoV-2 preimmunized mice, revealing an absence of immune imprinting, thus indicating its suitability for use in future sarbecovirus-origin epidemics or pandemics.

The widespread and rapid dissemination of antimicrobial resistance (AMR) is a global burden, and its consequences, especially in healthcare settings, are threatening. ESKAPE pathogens, a group of common nosocomial bacteria, have developed high levels of resistance to multiple antibiotics, leading to adverse effects, such as increased morbidity and mortality. As a result, there is an urgent need for effective strategies to combat AMR. Among the various available strategies, antimicrobial peptides (AMPs) have emerged as a promising approach to deal with AMR. This review elaborates on the diverse nature of AMPs and their applications, with a particular focus on ESKAPE pathogens. It also provides an overview of the current status of AMP research including both in vitro and in vivo studies and explores their potential in combination therapies against drug-resistant pathogens. The current challenges and future prospects of using AMPs in a clinical setting have also been discussed to acknowledge the gap between laboratory research and clinical application.

Bacillus anthracis is the etiological agent of anthrax and is classified as a Tier 1 biothreat pathogen. The fluoroquinolone ciprofloxacin is a preferred prophylactic drug for potential anthrax infections and acts by stabilizing DNA strand breaks formed by the bacterial type II topoisomerases, gyrase and topoisomerase IV. Unfortunately, widespread fluoroquinolone usage has increased levels of resistance in common bacterial pathogens, raising concern that resistant B. anthracis strains could be misused. Therefore, there is great interest in developing new classes of antibacterials that are efficacious against both wild-type and fluoroquinolone-resistant B. anthracis infections. Previous studies have demonstrated that gepotidacin, a triazaacenaphthylene antibacterial that targets gyrase and topoisomerase IV, displays potent activity against B. anthracis and was efficacious in a rabbit inhalation anthrax model. Given these promising results, we evaluated the activity of OSUAB-0284, a Novel Bacterial Topoisomerase Inhibitor (NBTI) that shares a general pharmacophore with gepotidacin, against B. anthracis. OSUAB-0284 displayed activity against B. anthracis that was comparable to or better than gepotidacin. Both compounds displayed activity against fluoroquinolone-resistant cells. Gepotidacin and OSUAB-0284 increased levels of gyrase- and topoisomerase IV-mediated DNA single-stranded breaks and inhibited the overall catalytic activity of the two enzymes. Both compounds were also more potent than ciprofloxacin against wild-type gyrase and topoisomerase IV and maintained activity against fluoroquinolone-resistant enzymes. Finally, OSUAB-0284 displayed efficacy in a mouse model of inhalation anthrax. These results provide mechanistic underpinnings supporting the use of gepotidacin and OSUAB-0284 against B. anthracis and suggest that they may be potential candidates for the treatment of anthrax.

Parasitic protozoa exhibit a high demand for iron, with mitochondrial iron metabolism representing a vulnerable target for chemotherapeutic intervention. We recently demonstrated that mitochondrial targeting of the iron chelator deferoxamine (DFO) via triphenylphosphonium (TPP) conjugation enhances its antiparasitic efficacy. To expand upon this strategy, mitochondrially targeted derivatives of DFO and deferasirox (DFX) were synthesized and evaluated for their activity against important human parasites. The DFX derivative mitoDFX was effective against Trypanosoma spp. and Toxoplasma gondii with remarkable selectivity. The fact that mitoDFX is a promising anticancer agent, which is likely safe to use in the context of human health, highlights the potential for drug repurposing in parasitology. Structure–activity relationship (SAR) studies and iron distribution analyses in trypanosomes revealed that mitochondrial targeting of the compounds, rather than iron chelation per se, is the main driver of the antiparasitic effects, underscoring the critical role of phosphonium salts in bioactivity.