ACS Infectious Diseases最新文献

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.

Gyrase and topoisomerase IV, enzymes that play critical roles during DNA replication, are the targets of fluoroquinolones and other antibacterials. Gyrase removes positive supercoils that accumulate ahead of replication forks, while topoisomerase IV untangles daughter chromosomes. Although topoisomerase IV is an essential enzyme in most bacteria, some species, including Mycobacterium tuberculosis and Mycobacteroides abscessus, encode only gyrase. In these species, gyrase is the sole target of fluoroquinolones and is believed to assume the cellular functions of both type II topoisomerases. Although fluoroquinolones and emerging antibacterials such as spiropyrimidinetriones induce gyrase-mediated DNA cleavage, there is evidence that inhibition of gyrase function also plays a role in drug-induced cell death under some circumstances. Therefore, we examined the effects of moxifloxacin and ciprofloxacin (fluoroquinolones) and zoliflodacin (spiropyrimidinetrione) on the three catalytic activities presumably carried out by gyrase in mycobacteria: decatenation of tangled DNA, negative supercoiling of relaxed DNA, and relaxation of positive supercoils. Under all circumstances, lower concentrations of antibacterials were required to inhibit intermolecular DNA decatenation as compared to the intramolecular DNA relaxation or supercoiling functions of gyrase. Differences in drug potency could not be attributed solely to rates of individual reactions or the DNA substrates utilized. Rather, results suggest that the potency of gyrase inhibition by interfacial antibacterials is modulated by the topological state of the DNA and its specific interactions with gyrase. Whereas most studies focus on DNA cleavage induced by gyrase-targeted antibacterials, this study provides mechanistic insights into how antibacterials rob replicating cells of essential gyrase functions.

Innate immune cells, such as monocytes and macrophages, provide the earliest defense against intracellular pathogen infection by initiating signaling pathways and restricting pathogen replication. However, the full complement of proteins that mediate cell-autonomous immunity remains incompletely defined. Here, we applied cysteine-directed activity-based protein profiling (ABPP) to map proteome-wide cysteine reactivity changes in THP-1 monocytes and primary human monocyte-derived macrophages during Mycobacterium tuberculosis (Mtb) infection. Across both cell types, we quantified 148 cysteine residues with altered reactivity. Knockdown of a subset of proteins harboring infection-induced reactivity significantly altered Mtb replication in THP-1 monocytes, linking proteins with reactive cysteines to antimicrobial defense. These data define previously unrecognized host protein changes during Mtb infection and provide a resource for investigating post-translational events that regulate innate immune responses to intracellular bacteria.

The development of innovative therapeutics against WHO priority pathogens is an urgent global need. Here, we demonstrate the functionalities of a human antibody, previously isolated by whole cell biopanning of the phage display scFv library. The yPgi3G4 IgG1 could mediate antibody-dependent phagocytosis of Pseudomonas aeruginosa by human matured THP-1 monocytes, of which colocalization with caveolin was clearly observed at 24 h. Next, the antibacterial activity against live P. aeruginosa was demonstrated by an agglutination assay and complement-mediated killing of the bacteria. Lipopolysaccharide (LPS) extraction and Western blot (WB) analysis suggested that LPS was the target of the yPgi3G4 IgG1 antibody. A cross-reactivity assay to available isolates in Thailand and France showed that the antibody could detect 6 P. aeruginosa serotypes including several multidrug-resistant clinical isolates. This study proved the potential of using this strategy to identify a biotherapeutic for a certain quotient of multidrug-resistant P. aeruginosa and other bacterial infections.

Malaria remains a major global health threat, and the emergence of partial artemisinin resistance challenges current treatment regimens. Reliable antimalarial screening assays are therefore essential for identifying new drug candidates. The parasite reduction ratio (PRR) assay provides valuable pharmacodynamic insights but is limited by its labor-intensive, 14- to 28-day incubation period. We developed an optimized PRR assay protocol using the highly sensitive chemiluminescence-based lacZ/β-gal^SENSOR readout, reducing assay incubation duration to 7 days while maintaining informative pharmacodynamic parameters, including lag phase, parasite clearance time, parasite reduction ratio, and maximum killing effect. In contrast, the [³H]-hypoxanthine incorporation method failed to detect viable parasites reliably and consistently overestimated drug activity with the shortened protocol. This novel lacZ/β-gal^SENSOR PRR assay enables laboratories without access to radioactive facilities to evaluate antimalarial compounds efficiently, providing robust time-killing profiles with greater convenience, higher throughput, and lower equipment requirements than existing readout methods.

The alphaviruses chikungunya (CHIKV) and Mayaro (MAYV) are responsible for acute febrile illnesses often accompanied by severe and persistent joint and muscle pain. Due to the lack of specific treatment, research into antivirals against these emerging viruses is seen as an urgent need. Previous studies demonstrated that silymarin exhibits potent antiviral activity against CHIKV and MAYV. Then, given the promising antiviral profile of silymarin, and the prominent joint and muscle pain caused by these viruses, we evaluated whether silymarin could reverse these damages in a murine model of alphavirus-induced arthritis and myositis. BALB/c mice were infected with CHIKV or MAYV in the right hind paw pad, and treated groups received silymarin orally (200 mg/kg/day). Clinical observation revealed reduced paw edema in silymarin-treated animals. At 7 and 12 days postinfection (dpi), animals were euthanized and various tissues collected. In infected and treated animals, a greater than 90% reduction in CHIKV viral load was observed in the spleen (7 and 12 dpi), paw (7 dpi), soleus muscle, and liver (12 dpi). Similarly, for MAYV, a greater than 90% reduction in viral load was detected in the spleen (7 and 12 dpi), liver, quadriceps, soleus muscle (7 dpi), and paw (12 dpi). Histological analysis revealed reduced inflammatory infiltrates in the liver, paw, and muscles as well as a decrease in both the number and area of lymphoid nodules in the spleen (12 dpi). Furthermore, silymarin treatment reduced TNF-α levels by at least 2-fold in the paw (7 and 12 dpi) and quadriceps (12 dpi). These findings suggest that silymarin not only limits viral replication in key target tissues, including the spleen, liver, muscle, and paw, but also mitigates inflammation by reducing paw edema, inflammatory infiltrates in hepatic, musculoskeletal, and paw tissues, the number and area of lymphoid nodules in the spleen, and TNF-α levels in the quadriceps muscle and paw, thereby supporting its therapeutic potential against CHIKV and MAYV infections.

Leprosy remains an important neglected tropical disease with about 200,000 new cases detected annually worldwide. Although the disease is highly responsive to treatment, a timely and accurate diagnosis continues to be a critical barrier to disease control. Traditional diagnostic methods, including PCR, bacilloscopy, histopathology, and serology, are hindered by limited sensitivity, procedural complexity, and restricted accessibility in resource-constrained settings. This review summarizes studies from the past decade on biosensor-based strategies for leprosy diagnosis. Biosensor platforms for leprosy include electrochemical, piezoelectric, and optical systems, with recent innovations encompassing immunosensors, biomimetic, and DNA-based approaches, some achieving diagnostic accuracies above 90%. These platforms employ different bioreceptors such as conjugated peptides, DNA probes, and molecularly imprinted polymers. Certain platforms can also differentiate paucibacillary from multibacillary cases, addressing a critical limitation of the current methods. These capabilities highlight the potential of biosensors as powerful tools for point-of-care testing. However, clinical translation is constrained by challenges such as affordability, robustness under field conditions, and the lack of large-scale validation studies. Additional operational barriers, including regulatory approval, supply chain logistics, and user training, must also be addressed. Future progress will depend on multidisciplinary strategies, integrating novel biomarker discovery as recognition elements and exploring detection systems previously used for other mycobacterial and infectious diseases. Large multicenter trials and user-centered design approaches are essential for clinical implementation. By overcoming these challenges, biosensors have the potential to redefine leprosy diagnostics, enabling earlier detection and improved surveillance, and accelerating progress toward global elimination goals.

Visceral leishmaniasis (VL) is a neglected tropical disease affecting humans and dogs, particularly in urban settings. Current therapies are limited by toxicity, lengthy regimens, and emerging drug resistance. No human vaccine is available, and only a few licensed formulations exist for canine use. Here, we evaluated a recombinant Leishmania infantum lipophosphoglycan-3 (rLPG3) antigen formulated with Freund's incomplete adjuvant (FIA) against Leishmania infantum challenge in BALB/c mice. The formulation reduced hepatic parasitism, increased antioxidant enzyme activities (superoxide dismutase, catalase, glutathione S-transferase), and raised total antioxidant capacity and hepatic nitrite/nitrate, while lipid and protein oxidation markers remained unchanged. Vaccination preserved liver architecture, lowered AST/ALT, reduced granuloma number and area, and shifted granuloma maturation toward organized lesions with greater macrophage content; PAS staining indicated higher hepatocyte glycogen in the rLPG3+FIA group. Serologically, rLPG3+FIA increased IgG1 and the IgG1/IgG2a ratio, indicating a Th2-skewed profile concomitant with reduced parasitism. Within the constraints of this model, time point, and the proof-of-concept use of FIA, these convergent readouts support rLPG3 as a promising antigen for further preclinical development─prioritizing licensable veterinary adjuvants to enable translation into canine VL vaccines.