ACS Infectious Diseases最新文献

Targeting the specific RNA conformations that are crucial for SARS-CoV-2 replication is a viable antiviral approach. The SARS-CoV-2 genome contains GG repeats capable of forming unstable two-tetrad G-quadruplex (GQ) structures, which exist as a mix of conformations, including hairpin (Hp), intra-, and intermolecular GQs. RGQ-1, originating from the nucleocapsid gene's ORF, adopts a dynamic equilibrium of conformations, including intramolecular hairpin and G-quadruplex (Hp-GQ) structures, as confirmed by CD analysis. In this study, tetraphenylethene (TPE) derivatives were developed to target the Hp-GQ conformational equilibrium of RGQ-1. EMSA, fluorescence spectroscopy, and ITC assays confirmed that two TPE derivatives, TPE-MePy and TPE-Allyl Py, bind to RGQ-1. CD thermal melting experiments indicate that RGQ-1 is stabilized by 8.56 and 12.54 °C in the presence of TPE-MePy and TPE-Allyl Py, respectively. Additionally, luciferase assays demonstrated that TPE derivatives suppressed luciferase activity by 2.2-fold and 3.6-fold, respectively, shifting the HpGQ equilibrium toward the GQ conformation, as suggested by CD spectroscopy. Treatment of SARS-CoV-2-infected A549 cells with TPE derivatives reduced the levels of viral RNA, spikes, and nucleocapsid proteins. To explore their antiviral mechanism, preinfection and postinfection treatments were tested, revealing that the TPE derivatives specifically suppressed the postentry stages of viral replication without affecting viral entry. These findings highlight the therapeutic potential of TPE derivatives in inhibiting key gene expressions critical for SARS-CoV-2 replication.

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant concern for skin and soft tissue infections. Apart from biofilm formation, these bacteria can reside intracellularly in phagocytic and nonphagocytic mammalian cells, complicating treatment with conventional antibiotics. Lipid-polymer hybrid nanoparticle (LPN) systems, combining the advantages of polymeric nanoparticles and liposomes, represent a new generation of nanocarriers with the potential to address these therapeutic challenges. In this study, gentamicin (Gen) and vancomycin (Van) were encapsulated in LPNs and evaluated for their ability to eliminate intracellular MRSA in phagocytic macrophage RAW-Blue cells and nonphagocytic epithelial HaCaT cells. Compared to free antibiotics at 100 μg/mL, LPN formulations significantly reduced intracellular bacterial loads in both cell lines. Specifically, LPN-Van resulted in approximately 0.7 Log CFU/well reduction in RAW-Blue cells and 0.3 Log CFU/well reduction in HaCaT cells. LPN-Gen showed a more pronounced reduction, with approximately 1.26 Log CFU/well reduction in RAW-Blue cells and 0.45 Log CFU/well reduction in HaCaT cells. In vivo, LPN-Van at 500 μg/mL significantly reduced MRSA biofilm viability compared to untreated controls (p < 0.001), achieving 98% eradication based on median values. In comparison, free vancomycin achieved a nonstatistically significant 79.2% reduction in biofilm viability compared to control. Prophylactically, LPN-Van at 500 μg/mL decreased MRSA levels to the limit of detection, resulting in a ∼3.5 Log reduction in the median CFU/wound compared to free vancomycin. No acute dermal toxicity was observed for LPN-Van based on histological analysis. These data indicate that LPNs show promise as a drug delivery platform technology to address intracellular infections.

This study delves into a newly discovered MBL (metallo-β-lactamase) in Clostridioides difficile, a formidable pathogen known for causing nosocomial infections and exhibiting resistance to antimicrobial agents. The primary objective was to unravel its structure-function relationship. This research establishes the enzyme AHM-1 as a subclass B3-like MBL. Experimental results reveal that the enzyme's active site consists of two Zn²⁺ atoms exhibiting tetrahedral and trigonal bipyramidal coordination, similar to B1 and B3 MBLs. Notably, within its active site, it exhibits a lower binding capacity for other transition metal ions such as Fe²⁺, Mn²⁺, and Ni²⁺ compared to Zn²⁺. The zinc-binding sites of B1 and B3 MBLs contain strictly conserved His116-His118-His196 and Asp120-Cys221/His121-His263. The absence of all the conserved residues except His116, Asp120, and His121 in the Zn-binding site distinctly separates this enzyme from these two MBL subclasses. Conserved zinc binding motifs present in B1 and B3 MBLs are H-X-H-X-D and H-X-H-X-D-H, respectively. The presence of the H-X-D-X-D-H motif in the enzyme, similar to that in B3 enzymes, along with sequence and structural analysis, places this new enzyme closer to the enzymes belonging to the B3 subclass. This study also identifies the likely catalytic residues responsible for its β-lactamase activity, similar to B3 MBLs. In contrast to MBLs, this enzyme displays hydrolytic activity toward aztreonam. It also shows higher catalytic efficiency toward higher generation cephalosporins. This study thus underscores the significance of a novel enzyme with β-lactamase activity in Clostridioides difficile, highlighting its potential implications for clinical treatment due to its disparities from conventional MBLs.

Neglected tropical diseases such as Chagas disease, human African trypanosomiasis, leishmaniasis, and schistosomiasis have a significant global health impact in predominantly developing countries, although these diseases are spreading due to increased international travel and population migration. Drug repurposing with a focus on increasing antiparasitic potency and drug-like properties is a cost-effective and efficient route to the development of new therapies. Here we identify compounds that have potent activity against Trypanosoma cruzi and Leishmania donovani, and the latter were progressed into the murine model of infection. Despite the potent in vitro activity, there was no effect on parasitemia, necessitating further work to improve the pharmacokinetic properties of this series. Nonetheless, valuable insights have been obtained into the structure-activity and structure-property relationships of this compound series.

Understanding bacterial physiology in real-world environments requires noninvasive approaches and is a challenging yet necessary endeavor to effectively treat infectious disease. Bacteria evolve strategies to tolerate chemical gradients associated with infections. The DIVER (Deep Imaging Via Enhanced Recovery) microscope can image autofluorescence and fluorescence lifetime throughout samples with high optical scattering, enabling the study of naturally formed chemical gradients throughout intact biofilms. Using the DIVER, a long fluorescent lifetime signal associated with reduced pyocyanin, a molecule for electron cycling in low oxygen, was detected in low-oxygen conditions at the surface of Pseudomonas aeruginosa biofilms and in the presence of fermentation metabolites from Rothia mucilaginosa, which cocolonizes infected airways with P. aeruginosa. These findings underscore the utility of the DIVER microscope and fluorescent lifetime for noninvasive studies of bacterial physiology within complex environments, which could inform on more effective strategies for managing chronic infection.

The secreted Chorismate mutase enzyme of Mycobacterium tuberculosis (*MtbCM) is an underexplored potential target for the development of new antitubercular agents that are increasingly needed as antibiotic resistance rises in prevalence. As an enzyme suspected to be involved in virulence and host-pathogen interactions, disruption of its function could circumvent the difficulty of treating tuberculosis-infected granulomas. Drug development, however, is limited by novel ligand discovery. Currently, *MtbCM activity is measured by using a low throughput acid/base-mediated product derivatization absorbance assay. Here, we utilized an RNA-display affinity selection approach enabled by the Random Peptides Integrated Discovery (RaPID) system to screen a vast library of macrocyclic peptides (MCP) for novel *MtbCM ligands. Peptides identified from the RaPID selection, and analogs thereof identified by analyzing the selection population dynamics, produced a new class of *MtbCM inhibiting MCPs. Among these were two noteworthy "chorismides", whose binding modes were elucidated by X-ray crystallography. Both were potent inhibitors of the CM enzyme activity. One was identified as an allosteric binding peptide revealing a novel inhibition approach, while the other is an active-site binding peptide that when conjugated to a fluorescent probe allowed for the development of a series of alternative fluorescence-based ligand-displacement assays that can be utilized for the assessment of potential *MtbCM inhibitors.

Malaria, caused by a protozoan parasite of the genus Plasmodium, is a severe infectious disease with life-threatening consequences that has burdened mankind for centuries. Although Plasmodium falciparum (P. falciparum) malaria is more prevalent globally than Plasmodium vivax (P. vivax) malaria, India bears the largest burden of P. vivax malaria, with over 3.6 million cases accounting for ∼48% of global P. vivax malaria cases. Existing detection methods for P. vivax malaria are costly or tedious or have low accuracy. To address the need for a specific diagnostic assay for P. vivax, we generated aptamers specific to Plasmodium vivax tryptophan-rich antigen (PvTRAg). We employed them in an aptamer-linked immobilized sorbent assay (ALISA) to detect P. vivax malaria infections. The two most specific aptamers for PvTRAg, identified as Apt_14 and Apt_16, were obtained using the Systematic Evolution of Ligands by Exponential Enrichment. The dissociation constant (K_D) values of Apt_14 and Apt_16 were 1.9 and 1.2 nM, respectively, indicating high affinity to PvTRAg. The limit of detection for both aptamers was found to be 2.5 nM. During clinical validation, the sensitivity of 96% and 84% was obtained with Apt_14- and Apt_16-based ALISA with 100% specificity. The aptamers demonstrated nonsignificant cross-reactivity with other nonmalarial antigens and PvTRAg homologues along with a high level of selectivity for PvTRAg over P. falciparum antigens and various other antigens. Altogether, our findings confirm the effectiveness of DNA aptamers for the accurate diagnosis of P. vivax malaria and lay the groundwork for developing an aptamer-based diagnostic assay for malaria.

Cryptococcosis is a severe fungal infection primarily caused by two encapsulated yeasts: Cryptococcus neoformans and C. gattii. The most significant complication is cryptococcal meningitis, where the fungus crosses the blood-brain barrier, leading to a severe brain infection. Current treatments, which include amphotericin B and flucytosine or fluconazole, are often toxic and not very effective. Therefore, there is a pressing need for new antifungal agents. This study screened 30 thiazole derivatives for their antifungal activity against Cryptococcus and their toxicity to brain cells. Four compounds (RN86, RN88, RJ37, and RVJ42) showed particularly strong effects. These compounds reduced ergosterol levels in the fungal membrane and inhibited its ability to cross the blood-brain barrier. Notably, RN86 and RVJ42 improved survival rates in a mouse model of cryptococcosis by lowering the fungal load in the lungs and brain. These findings suggest that these derivatives could be promising treatments for pulmonary and neurocryptococcosis.

The integration of RNA- and DNA-based assays enables the investigation of disease dynamics, specifically assessing the role of asymptomatic or subclinical infections in malaria transmission. Circular RNAs (circRNAs), a distinct category of noncoding RNAs, are implicated in numerous pathogenic mechanisms. As of now, research has yet to explore circRNAs' function in malaria infection. The findings revealed that Plasmodium infection upregulated 60 circRNAs and downregulated 71 in BALB/c mice. We selected 11 differentially expressed (DE) circRNAs according to function prediction of target miRNA-mRNA and coding protein, and these were further confirmed by validation experiments. IRESfinder, GO, and KEGG evaluations indicated that 7 DE circRNAs possess protein-coding potential and are enriched in the MAPK signaling cascade. In P.y17XL-infected BALB/c mouse models, the findings substantiated that the dynamic characteristics of DE circRNAs correlated with inflammation, and the MAPK and NF-κB signaling cascades were activated, also contributing to the inflammatory reaction during malaria infection. This study establishes Plasmodium-induced circRNA expression as a novel mechanism by which the parasite modulates host immune signaling, advancing the understanding of Plasmodium-host cell interactions. In addition, 42 circRNAs were found in normal BALB/c mice, and 25 circRNAs were discovered in P.y17XL-infected BALB/c mice, excluding 1238 circRNAs shared by normal and P.y17XL-infected BALB/c mice. Plasmodium infection changes the expression profile of circRNAs in the host, and these altered circRNAs are involved in the inflammatory response during malaria infection. In addition, Plasmodium possibly regulates the reverse splicing of pre-mRNA or m6A modification of RNA, inducing the production of novel circRNAs in the host.

Nonstructural protein 1 of influenza A (NS1A) is a key virulence factor produced inside host cells infected with Influenza A Virus (IAV) and consists of an N-terminal dsRNA binding domain (RBD) and a C-terminal effector domain (ED), joined by a flexible linker. While NS1A is a highly promiscuous protein with a number of intracellular functions, its primary function is nonspecific dsRNA binding that enables influenza to evade our innate immune system. For this reason, NS1A has long been proposed as a potential drug target. Previous research in the field has demonstrated the necessity of dimer formation through the RBD to enable dsRNA binding, which is further enhanced by oligomerization through ED interactions. However, there has been minimal exploration of potential strain-specific effects on dsRNA binding. Most existing studies are limited to the A/Udorn/307/1972 strain, often with a C-terminal tail deletion. Here we utilize fluorescence polarization (FP) paired with fluorescence-based electrophoretic mobility shift assays (fEMSA) to characterize the dsRNA binding properties of NS1A from the H1N1 strain responsible for the 1918 "Spanish Flu" with an intact C-terminal tail. We show that A/Brevig Mission/1/1918 NS1A contains specific residues in the RBD that enhance dsRNA binding. We further demonstrate that both Brevig Mission and Udorn NS1A bind directly to dsRNA through the highly basic C-terminal tail of the ED. These novel binding interactions may have contributed to the increased pathogenicity of the 1918 flu pandemic and may have implications for NS1A-targeted antivirals.