ACS Infectious Diseases最新文献

The nontuberculous mycobacterium (NTM) Mycobacterium abscessus (Mab) has emerged as a global health concern due to its high intrinsic resistance toward antibiotics. The search for anti-NTM inhibitors requires novel well-characterized targets. The cytochrome bd (cyt-bd) oxidase, which serves as an alternate terminal oxidase in mycobacteria, is a chemically validated drug target in Mycobacterium tuberculosis (Mtb). However, no genetic, biochemical, or structural studies have been described for the Mab enzyme. Successful targeting of the Mab cyt-bd oxidase requires an in-depth understanding of its mechanistic and regulatory elements. Here, we generated a homology model of Mab cyt-bd, including the alternate menaquinol-binding pocket, the predicted oxygen channel, the proposed redox modulation site (C266-C285), and the salt bridge pair, keeping the cysteine residues in proximity. A heterologous system was developed for whole-cell functional studies to characterize the impact of mutations in these critical domains on enzyme activity. Mutating W9, E98, F103, or E263 to alanine inhibited the enzyme totally, underscoring their importance in menaquinol binding, oxygen reduction, and/or redox modulation. The Mab cyt-bd C285A mutant displayed a reduction in oxygen consumption and ATP formation, a phenomenon also presented for the Mtb C285A mutant. In summary, this study presents the first structural and biochemical characterization of Mab cyt-bd oxidase, providing insights into the importance of mechanistic and regulatory elements of the Mab enzyme in a whole-cell setup, which will be of relevance for the design of anti-NTM and antituberculosis hit molecules targeting this oxidase.

We observed a high proportion of proteins in pathogenic Mycobacterium species that can potentially undergo liquid-liquid phase separation (LLPS) mediated biomolecular condensate formation, compared to nonpathogenic species. These proteins mainly include the PE-PPE and PE-PGRS families of proteins that have nucleic acid and protein-protein binding functions, typical of LLPS proteins. We also mapped identified LLPS proteins in M. tuberculosis (M.tb) drug-resistant databases PubMLST and TBProfiler, based upon the WHO 2023 catalogue of resistance-associated mutations. High sequence conservation of LLPS-associated proteins in various multiple drug-resistant M.tb isolates points to their potentially important role in virulence and host-pathogen interactions during pathogenic evolution. This analysis provides a perspective on the role of protein phase separation in the evaluation of M.tb pathogenesis and offers avenues for future research aimed at developing innovative strategies to combat M.tb infection.

The rise of multidrug-resistant tuberculosis (TB) has increased the need for new antitubercular (anti-TB) drugs and the identification of novel drug targets. One promising target is Mycobacterium tuberculosis (Mtb) cytochrome P450 enzymes (P450s). This study focuses on the characterization of CYP135B1, a prevalent Mtb P450. Using a combination of microbiology, genomics, bioinformatics, docking, spectroscopy, and mass spectrometry, researchers successfully expressed, purified, and characterized CYP135B1. A 3D model was built with AlphaFold 3. The enzyme displayed typical features of P450 proteins and showed strong binding to imidazole derivatives. Notably, CYP135B1 metabolized the anti-TB drug SQ109 by inserting oxygen into its geranyl moiety in a manner distinct from CYP124A1. However, genetic studies using a ΔCYP135B1 mutant strain revealed that CYP135B1 is not required for SQ109's antibacterial activity, as its deletion did not affect drug efficacy despite CYP135B1 metabolizes SQ109.

Mitochondria are important organelles that regulate energy homeostasis. Mitochondrial health and dynamics are crucial determinants of the outcome of several bacterial infections. SIRT3, a major mitochondrial sirtuin, along with SIRT1 regulates key mitochondrial functions. This led to considerable interest in understanding the role of SIRT1 and SIRT3 in governing mitochondrial functions during Salmonella infection. Here, we show that loss of SIRT1 and SIRT3 function either by shRNA-mediated knockdown or by inhibitor treatment led to increased mitochondrial dysfunction with alteration in mitochondrial bioenergetics alongside increased mitochondrial superoxide generation in Salmonella-infected macrophages. Consistent with dysfunctional mitochondria, mitophagy was induced along with altered mitochondrial fusion-fission dynamics in S. typhimurium-infected macrophages. Additionally, the mitochondrial bioenergetic alteration promotes acidification of the infected macrophage cytosolic pH. This host cytosolic pH imbalance skewed the intraphagosomal and intrabacterial pH in the absence of SIRT1 and SIRT3, resulting in decreased SPI-2 gene expression. Our results suggest a novel role for SIRT1 and SIRT3 in maintaining the intracellular Salmonella niche by modulating the mitochondrial bioenergetics and dynamics in the infected macrophages.

Treatment of Mycobacterium abscessus lung disease relies on underperforming drug combinations and includes parenteral, poorly tolerated, and bacteriostatic antibiotics. We posit that safe, oral, and bactericidal regimens are needed to improve cure rates and shorten treatment. Here, we combined oral representatives of three well-tolerated bactericidal drug classes, the β-lactam tebipenem (together with the β-lactamase inhibitor avibactam), the fluoroquinolone moxifloxacin, and the rifamycin rifabutin, and profiled the combination in vitro and in vivo. The combination potentiated bactericidal activity of its components against replicating M. abscessus and retained bactericidal activity against the nonreplicating, drug-tolerant form of the bacterium residing in surrogate caseum. When combined, the drugs retained the ability to induce lethal secondary effects associated with the β-lactam and fluoroquinolone, including cell wall and DNA damage, increased metabolism, and generation of reactive oxygen species. Thus, the triple-drug combination appears to exert two lethal punches while suppressing bacterial reprogramming to counter the drug-induced stresses, providing a plausible rationale for the enhanced kill effect. Addition of a bacteriostatic agent resulted in drug-specific patterns of interactions with regards to bactericidal activity reflected by the lethal secondary effects. The triple-drug combination also exerted a pronounced postantibiotic effect and reduced emergence of spontaneous resistant mutants. Collectively, this work provides a combination prototype for optimization and a profiling workflow that may be useful for the development of sterilizing regimens.

The postantibiotic effect (PAE) is the persistent suppression of microbial growth following the removal of antimicrobial therapy. In general, antibiotics that generate a PAE are dosed less frequently, and thus, the PAE has important implications for dosing regimens. PAEs can arise through several mechanisms, including the extended occupancy of the drug target following drug elimination, and the correlation between drug-target residence time and PAE provides insight into target vulnerability. To assess the vulnerability of Escherichia coli leucyl-tRNA synthetase (ecLeuRS), which is an essential enzyme in protein synthesis, the time-dependent inhibition of the enzyme was studied by the benzoxaborole class of compounds that inhibit LeuRS by forming a stable LeuRS-tRNA^Leu-benzoxaborole adduct. Preincubation of epetraborole with ecLeuRS resulted in a decrease in the IC₅₀ value for enzyme inhibition from 38 to 3 nM, consistent with the slow formation of the final enzyme-inhibitor complex, and similar shifts in IC₅₀ were observed for three other benzoxaboroles. The benzoxaboroles generated short PAEs (<1 h) in E. coli, however, the PAE values of AN3334 and epetraborole increased from 0.88 to 1.70-3 h when a sub-MIC concentration of the aminoglycoside tobramycin was included in the media. pSILAC revealed that the synthesis rate of ecLeuRS was reduced 1.6-fold in the presence of sub-MIC tobramycin, reinforcing the role that protein turnover plays in target vulnerability.

New antifungals are urgently needed to treat deadly fungal infections. Targeting the fungal inositol polyphosphate kinases IP_3-4K (Arg1) and IP₆K (Kcs1) is a promising strategy as it has been validated genetically to be crucial for fungal virulence but never pharmacologically. We now report the synthesis of DT-23, an analogue of N2-(m-trifluorobenzylamino)-N6-(p-nitrobenzylamino)purine (TNP), and demonstrate that it more potently inhibits recombinant Arg1 from the priority pathogen Cryptococcus neoformans (Cn) (IC₅₀ = 0.6 μM) than previous analogues (IC₅₀ = 10-30 μM). DT-23 also inhibits recombinant Kcs1 with similar potency (IC₅₀ = 0.68 μM) and Arg1 and Kcs1 activity in vivo. Unlike previous analogues, DT-23 inhibits fungal growth (MIC₅₀ = 15 μg/mL) and only 1.5 μg/mL synergizes with Amphotericin B to kill Cn in vitro. DT-23/Amphotericin B is also more protective against Cn infection in an insect model compared to each drug alone. Transcription profiling shows that DT-23 impacts early stages in IP synthesis and cellular functions impacted by IPK gene deletion, consistent with its targeted effect. This study establishes the first pharmacological link between inhibiting IPK activity and antifungal activity, providing tools for studying IPK function and a foundation to potentially develop a new class of antifungal drug.

Innovative antimalarials are required to combat malaria, a global infectious disease caused by Plasmodium falciparum. To explore the untapped antiplasmodial compounds that can target the iron source vital at the blood stages of P. falciparum, we investigated the antiplasmodial activities of natural siderophores and synthetic compounds with metal-binding affinity. The assessment of their IC₅₀ values and spectroscopic analytical data revealed that terpyridyl compounds specifically bound to target Fe(II) ions and strongly induced the growth inhibition of intraerythrocytic parasites. Furthermore, the IC₅₀ values of the 4,4',4''-substituted terpyridines were linearly correlated with the sum of the para Hammett constants of their substitutions, suggesting that their growth inhibitory effects depended on the electronic states of the coordinating nitrogen atoms. Considering the specific developmental blockage at the trophozoite stage and selective antiplasmodial activities of the iron-grabbing compounds, these findings provide insights into the development of antimalarials that can disrupt iron homeostasis.

Arthrospira platensis, an ancient cyanobacterium, is rich in bioactive compounds with therapeutic potential, supporting its use in studies for various health conditions, including infectious and chronic diseases. This study aimed to evaluate the antiparasitic, cytotoxic, and immunomodulatory activities of A. platensis compounds against Trypanosoma cruzi. Peripheral Blood Mononuclear Cells (PBMC) and T. cruzi trypomastigotes were cultured for cytotoxic and antiparasitic analyses. Cytotoxicity was assessed in PBMC treated with different concentrations of crude extract, obtained by mechanical agitation in 0.1 M TRIS-HCl buffer (pH 7.2), and purified protein by DEAE-Sephadex A-50 chromatography and FPLC. Immune response was analyzed in infected and uninfected PBMC by measuring cytokines (IFN-γ, TNF, IL-2, IL-6, and IL-10) after treatment with purified protein and benznidazole. In vitro experiments showed that both crude extract and a 15 kDa purified protein were toxic to trypomastigotes in a dose-dependent manner, eliminating over 80% of the parasite at 1000 and 200 μg/mL, respectively. Both the extract and protein were nontoxic to PBMC, with the protein (SI: 20.7) being more selective than benznidazole (SI: 11.5). Results indicated that the purified protein modulated the immune response in T. cruzi-infected individuals, inducing a protective Th1 response while controlling an excessive inflammatory response with appropriate IL-10 levels. The anti-T. cruzi activity of this protein, alone or in combination with the commercial drug, suggests it could be a low-cost, safer, and more tolerable therapy for Chagas disease treatment.