ACS Infectious Diseases最新文献

Penicillin-binding proteins (PBPs) are an essential family of bacterial enzymes that are covalently inhibited by the β-lactam class of antibiotics. PBP inhibition disrupts peptidoglycan biosynthesis, which results in deficient growth and proliferation, and ultimately leads to lysis. IC₅₀ values are often employed as descriptors of enzyme inhibition and inhibitor selectivity, but can be misleading in the study of time-dependent, covalent inhibitors. Due to this disconnect, the second-order rate constant, k_inact/K_I, is a more appropriate metric of covalent-inhibitor potency. Despite being the gold standard measurement of potency, k_inact/K_I values are typically obtained from in vitro assays, which limits assay throughput if investigating an enzyme family with multiple homologues (such as the PBPs). Therefore, we developed a whole-cell k_inact/K_I assay to define inhibitor potency for the PBPs in Streptococcus pneumoniae using the fluorescent, activity-based probe, Bocillin-FL. Our results align with in vitro k_inact/K_I data and show a comparable relationship to previously established IC₅₀ values. These results support the validity of our in vivo k_inact/K_I method as a means of obtaining β-lactam potency for a suite of PBPs to enable structure-activity relationship studies.

Recently, a high-level daptomycin (DAP)-resistant Mammaliicoccus sciuri strain (TS92) was identified, which mediates a 33% decline of DAP when incubated in Mueller-Hinton (MH) medium. The genetic background of the DAP resistance in TS92 is a newly discovered two-gene operon, named drcAB, whose expression was reported to impair the structural integrity of DAP, eventually leading to its inactivation. Here, we set out to elucidate the chemical nature of drcAB-mediated DAP modification by applying a general unknown comparative screening (GUCS) approach in high-resolution mass spectrometry. DAP in MH medium was incubated with Staphylococcus aureus strain RN4220_P_xyl/tet-drcAB, which carries the drcAB operon under control of an inducible promoter on a plasmid, and GUCS test and reference samples were obtained upon and without drcAB expression. A two-step process catalyzed by DrcAB was discovered, comprising a structural alteration of DAP. The mass spectrometric data indicate an N-substitution at the aniline moiety of kynurenine with dehydroalanine and, subsequently, a cleavage of the ester bond of the DAP core between kynurenine and threonine by means of water. The structures postulated were confirmed by comparison of in silico versus measured fragmentation patterns.

Chronic Chagas cardiomyopathy is associated with an unbalanced immune response and impaired heart function, and available drugs do not prevent its development. Zileuton (Zi), a 5-lypoxigenase inhibitor, affects inflammatory/pro-resolution mediators. Herein, Zi treatment in the early phase of infection reduced parasitemia associated mainly with the direct effect of Zi on the parasite, and the enzyme epoxide hydrolase was the potential molecular target behind the trypanocidal effect. In the intermediate acute phase of infection, Zi reduced the number of innate and adaptive inflammatory cells, increased the level of SOCS2 expression in the heart associated with lower inflammation, and improved cardiac function. Zi treatment initiated in the chronic stage increased the level of SOCS2 expression in the heart, reduced inflammation, and improved cardiac function. Our data suggest that Zi protects against Trypanosoma cruzi infection by acting directly on the parasite and reducing heart damage and is a promising option for the treatment of Chagas disease.

The escalating prevalence of bacterial infections and the rapid emergence of multidrug-resistant Gram-negative bacterial pathogens highlight an urgent demand for effective antibacterial agents. In this study, we report our findings on IITR00210, a small molecule belonging to the nitrile class. The small molecule demonstrates broad-spectrum activity against bacterial pathogens, specifically against enteric pathogens, and exhibits antibiofilm activity. IITR00210 displays potent bactericidal activity against enteropathogens, resulting in a reduction of bacterial counts greater than 3 Log₁₀ CFU in time-kill kinetic assays. Mechanistic investigations revealed that IITR00210 induces bacterial cell envelope stress, leading to the alteration of the overall proton motive force (PMF). The disruption of PMF causes intracellular ATP dissipation and ultimately promotes cell death. The cell envelope stress generated in the presence of IITR00210 leads to a translational aberration. Importantly, IITR00210 exhibits a safe profile in in vitro and in vivo settings. The small molecule further showed potent intracellular antibacterial activity in polymorphonuclear cells infected with enteric pathogens and antiadhesion activity in mammalian cell lines. IITR00210 proves to be a promising therapeutic candidate, displaying a lack of stable resistance development, and it exhibited efficacy in the treatment of bacterial infections in a shigellosis murine model.

Antibiotic resistance in bacteria is a major global health concern. The wide spread of carbapenemases, bacterial enzymes that degrade the last-resort carbapenem antibiotics, is responsible for multidrug resistance in bacterial pathogens and has further significantly exacerbated this problem. Acinetobacter baumannii is one of the leading nosocomial pathogens due to the acquisition and wide dissemination of carbapenem-hydrolyzing class D β-lactamases, which have dramatically diminished available therapeutic options. Thus, new antibiotics that are active against multidrug-resistantA. baumannii and carbapenemase inhibitors are urgently needed. Here we report characterization of the interaction of the C5α-methyl-substituted carbapenem NA-1-157 with one of the clinically important class D carbapenemases, OXA-58. Antibiotic susceptibility testing shows that the compound is more potent than commercial carbapenems against OXA-58-producingA. baumannii, with a clinically sensitive MIC value of 1 μg/mL. Kinetic studies demonstrate that NA-1-157 is a very poor substrate of the enzyme due mainly to a significantly reduced deacylation rate. Mass spectrometry analysis shows that inhibition of OXA-58 by NA-1-157 proceeds through both the classical acyl-enzyme intermediate and a reversible covalent species. Time-resolved X-ray crystallographic studies reveal that upon acylation of the enzyme, the compound causes progressive decarboxylation of the catalytic lysine residue, thus severely impairing deacylation. Overall, this study demonstrates that the carbapenem NA-1-157 is highly resistant to degradation by the OXA-58 carbapenemase.

The secretory proteome of Plasmodium exhibits differential spatial and functional activity within host cells. Plasmodium secretes proteins that translocate into the human host cell nucleus. Liver-specific protein 2 of Plasmodium falciparum (Pf-LISP2) shows nuclear accumulation in human hepatocytes during the late liver stage of malaria parasite development. However, the nuclear translocation mechanism for Pf-LISP2 remains largely uncharacterized. Here, we identified a classical bipartite nuclear localization signal (NLS) located in the C-terminal region of Pf-LISP2. Phylogenetic analysis revealed that this NLS is unique to Plasmodium falciparum and its close relative Plasmodium reichenowi, suggesting an evolutionary adaptation linked to their shared primate hosts. Functional assays confirmed the NLS's nuclear import activity, as fusion constructs of the Pf-LISP2 NLS with Pf-aldolase (Pf-aldolase-NLS-EGFP) localized exclusively to the nucleus of HepG2 cells. Mutation analysis of key lysine and arginine residues in the bipartite NLS demonstrated that the basic amino acid clusters are essential for nuclear localization. Importin-α/β interaction was found to be crucial for Pf-LISP2 nuclear transport, as coexpression of the NLS constructs with the importin-α/β inhibitor mCherry-Bimax2 significantly blocked nuclear translocation. Specific interactions between the lysine and arginine residues of Pf-LISP2's NLS and the conserved tryptophan and asparagine residues of human importin-α1 facilitate the cytosol-to-nuclear translocation of Pf-LISP2. Additionally, LISP2 lacks any nuclear export signal. These results provide new insights into the mechanisms of nuclear transport in Plasmodium falciparum, potentially contributing to the understanding of its pathogenicity and host-cell interactions during liver-stage infection.

Enteroviruses cause significant morbidity and mortality worldwide, and Coxsackievirus B3 (CVB3) is one of the most commonly reported. Coxsackieviruses establish persistent infection, characterized as infection that is not cleared from host cells generating a continuous infection. No antivirals targeting persistent or acute infection are available, and CVB3 may respond differently depending on the type of infection. Therefore, there is an urgent need for new antiviral drugs to combat acute and persistent CVB3 infection. We developed a system to study persistent CVB3 infection with pancreatic ductal cell line PANC-1, and we used an epithelial cell line, Vero-E6 cells, to study acute CVB3 infection. We maintained persistently infected cells for over a year. Now, in an effort to identify antivirals, using the National Institutes of Health's Developmental Therapeutics Program (DTP), we screened thousands of compounds for activity against acute and persistent CVB3 infection, and among the hits was Ro 5-3335, a 1,4-benzodiazepine nordazepam that acts as a RUNX1-CBFβ leukemia inhibitor. Ro 5-3335 has previously been reported to inhibit HIV-1 gene expression through interference with Tat-mediated transactivation. We confirmed Ro 5-3335's antiviral activity against CVB3 in both acute and persistent infection, in several cell types and at pharmacologically favorable conditions. We show that Ro 5-3335 has minimal cytotoxicity and is antiviral over several rounds of replication. We identified viral egress as a putative target. We also show efficacy against other RNA viruses, but it is ineffective against a model DNA virus. Overall, Ro 5-3335 is a promising antiviral that may target CVB3 infection.

The proteasome is essential for eukaryotic cell proteostasis, and inhibitors of the 20S proteasome are progressing preclinically and clinically as antiparasitics. We screenedTrypanosoma brucei, the causative agent of human and animal African trypanosomiasis, in vitro with a set of 27 carmaphycin B analogs, irreversible epoxyketone inhibitors that were originally developed to inhibit thePlasmodium falciparum20S (Pf20S). The structure-activity relationship was distinct from that of the human c20S antitarget by the acceptance of d-amino acids at the P3 position of the peptidyl backbone to yield compounds with greatly decreased toxicity to human cells. For the three most selective compounds, binding to the Tb20S β5 catalytic subunit was confirmed by competition with a fluorescent activity-based probe. For one compound, J-80, with its P3 d-configuration, the differential binding to the parasite's β5 subunit was supported by both covalent and noncovalent docking analysis. Further, J-80 was equipotent against both Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense in vitro. In a mouse model of Stage 1 T. brucei infection, a single intraperitoneal (i.p.) dose of 40 mg/kg J-80 halted the growth of the parasite, and when given at 50 mg/kg i.p. twice daily for 5 days, parasitemia was decreased to below the detectable limit, with parasite recrudescence 48 h after the last dose. The in vivo proof of principle demonstrated by a potent, selective, and irreversible inhibitor of Tb20S reveals an alternative path to the development of kinetoplastid proteasome inhibitors that differs from the current focus on allosteric reversible inhibitors.

Shiga-toxin-producing Escherichia coli (STEC) are important human pathogens causing diarrhea, hemorrhagic colitis, and severe hemolytic uremic syndrome. Timely detection of the multifaceted STEC is of high importance but is challenging and labor-intensive. An easy-to-perform rapid test would be a tremendous advance. Here, the major STEC virulence factor Shiga toxins (Stx), RNA-N-glycosidases targeting the sarcin ricin loop (SRL) of 28S rRNA, was used for detection. We designed synthetic FRET-based ssDNA SRL substrates, which conferred a fluorescence signal after cleavage by Stx. Optimal results using bacterial culture supernatants or single colonies were achieved for substrate StxSense 4 following 30 to 60 min incubation. Stx1 and Stx2 subtypes, diverse STEC serotypes, and Shigella were detected. Within a proof-of-principle study, a total of 94 clinical strains were tested, comprising 65 STEC, 11 Shigella strains, and 18 strains of other enteropathogenic bacteria without Stx. In conclusion, the assay offers rapid and facile STEC detection based on a real-time readout for Stx activity. Therefore, it may improve STEC risk evaluation, therapy decisions, outbreak, and source detection and simplify research for antimicrobials.