The rapid emergence of antibiotic-resistant strains of presents a substantial challenge to global public health, underscoring the urgent need for novel antibiotics with diverse mechanisms of action. In this study, we conducted mutagenesis on the -terminal region of the lantibiotic ripcin C to enhance its antimicrobial efficacy against . The resulting optimized variant, ripcin C, demonstrated potent and selective antimicrobial activity, with a minimal inhibitory concentration of 2-4 mg/L against . Beyond its strong antimicrobial properties, ripcin C exhibited significant antibiofilm activity against methicillin-resistant (MRSA). Mechanistic studies revealed that, in addition to targeting lipid II, ripcin C disrupts bacterial membranes, a capability absent in ripcin C, which may contribute to its superior antimicrobial and antibiofilm effects. Moreover, ripcin C displayed favorable biosafety and plasma stability profiles. Notably, in a mouse model of MRSA-induced mastitis, ripcin C effectively reduced bacterial load, alleviated inflammation, and preserved the normal histomorphology of mammary glands. This study introduces ripcin C as a promising antibiotic candidate for the treatment of MRSA-related infections.

Well-tolerated and novel antimalarials that can combat multiple stages of the parasite life cycle are desirable but challenging to discover and develop. Herein, we report results for natural product-inspired novel tambjamine antimalarials. We show that they are potent against liver, asexual erythrocytic, and sexual erythrocytic parasite life cycle stages. Notably, our lead candidate (KAR425) displays excellent oral efficacy with complete clearance of parasites within 72 h of treatment in the humanized (NOD-scid) mouse model at 50 mg/kg × 4 days. Profiling of compound demonstrated a fast killing profile. In addition, several other tambjamine analogues cured erythrocytic infections after oral doses of 30 and 50 mg/kg × 4 days in a murine model while exhibiting good safety and metabolic profiles. This study presents the first account of multiple-stage antiplasmodial activities with rapid killing profile in the tambjamine family.

The escalating prevalence of bacterial infections presents a formidable challenge to current global healthcare systems. Rapid identification and quantification of bacterial pathogens with anticipated sensitivity and selectivity are crucial for targeted therapeutic interventions to mitigate disease burden, drug resistance, and further transmission. Concurrently, there is a pressing need to innovate novel approaches to combat infections and counter antibiotic resistance. Herein, we demonstrated the development of heparin (HP) conjugates modified with a Zn-induced "turn-on" fluorophore, 2-(pyridin-2-yl)-1-benzo[]imidazole (PBI), that interacts with bacterial cells via specific binding with the surface-exposed heparin-binding proteins (HPBs), thereby inducing fluorescence signals for rapid and selective sensing of whole bacterial cells. Additionally, amikacin (Amk) antibiotic was integrated into the modified heparin polymer (HP-PBI-Amk) to augment its antibacterial efficacy via reactive oxygen species generation. Despite the nephrotoxicity of only amikacin, its inclusion in the biopolymer retains its antibacterial properties while providing biocompatibility. The outcome of this study demonstrates the development of HP-PBI and HP-PBI-Amk as promising strategies for bacterial detection and eradication, respectively, offering potential avenues for future research and clinical applications.

The coronavirus pandemic outbreak of 2019 highlighted the critical importance of preparedness for current and future public health threats (https://www.mmv.org/mmv-open/global-health-priority-box/about-global-health-priority-box). While the main attention for the past few years has been on COVID-19 research, this focus has reduced global resources on research in other areas, including malaria and neglected tropical diseases (NTDs). Such a shift in focus puts at risk the hard-earned progress in global health achieved over the past two decades (https://www.who.int/news-room/spotlight/10-global-health-issues-to-track-in-2021). To address the urgent need for new drugs to combat drug-resistant malaria, emerging zoonotic diseases, and vector control, Medicines for Malaria Venture (MMV) and Innovative Vector Control Consortium (IVCC) assembled a collection of 240 compounds and, in August 2022, launched the Global Health Priority Box (GHPB). This collection of compounds has confirmed activity against emerging pathogens or vectors and is available free of charge. This valuable tool enables researchers worldwide to build on each other's work and save precious time and resources by providing a starting point for the further development of treatments and insecticides. Furthermore, this open access box aligns with two of the many priorities outlined by the World Health Organization (WHO) (https://www.who.int/news-room/spotlight/10-global-health-issues-to-track-in-2021).

In recent years, naturally occurring darobactins have emerged as a promising compound class to combat infections caused by critical Gram-negative pathogens. In this study, we describe the in vivo evaluation of derivative D22, a non-natural biosynthetic darobactin analogue with significantly improved antibacterial activity. We found D22 to be active in vivo against key critical Gram-negative human pathogens, as demonstrated in murine models of thigh infection, peritonitis/sepsis, and urinary tract infection (UTI). Furthermore, we observed the restored survival of -infected embryos in a zebrafish infection model. These in vivo proof-of-concept (PoC) in diverse models of infection against highly relevant pathogens, including drug-resistant isolates, highlight the versatility of darobactins in the treatment of bacterial infections and show superiority of D22 over the natural darobactin A. Together with a favorable safety profile, these findings pave the way for further optimization of the darobactin scaffold toward the development of a novel antibiotic.

In the chronic phase of Chagas disease (CCD), diagnosis relies on detecting specific IgG antibodies due to the low or absent presence of the parasite in human blood. However, the performance of current serological tests is highly variable, lacking a "" assay with 100% sensitivity and specificity, which challenges the exploration of new biomarkers. In the present study, we evaluated the diagnostic accuracy of an optimized ELISA using the predicted immunogenic domains (called TcD3 and TcD6) of Tc323, a protein highly conserved among strains but absent in other clinically significant parasites such as . This study was conducted using plasma or serum samples from CCD individuals with different clinical manifestations and living in endemic regions in Latin America, subjects with unrelated infectious diseases, and noninfected donors. The sensitivity and specificity of recombinant TcD3 were 90.8% and 92.6%, respectively, while rTcD6 displayed values of 93.1% and 93.6% for the same parameters. Area under curve (AUC) values were 0.949 for rTcD3 and 0.954 for rTcD6. The receiver operative characteristic (ROC) curve showed a highly significant difference between CCD individuals and noninfected donors. Cross-reactivity was 10.2% for rTcD3 and 8.2% for rTcD6 in subjects infected with leishmaniasis or with toxoplasmosis. In addition, the reactivity against rTcD3 differed among some geographical areas while no significant difference was found using both domains for the detection of -infected individuals with or without cardiac symptoms. Our findings show that the recombinant antigens rTcD3 and rTcD6 could be used as highly potential biomarkers for the serological diagnosis of CCD.

Intracellular parasites, including and , are entirely reliant on the active scavenging of host-derived nutrients to fuel their replicative cycle, as they are confined within a specialized membrane-bound compartment, the parasitophorous vacuole (PV). Initial observations, based on the proximity of host vesicles to the parasitophorous vacuole membrane (PVM), suggested that parasites utilize host vesicles to obtain essential nutrients. However, mounting evidence has now unequivocally demonstrated that intracellular pathogens establish membrane contacts with host organelles, establishing control over host cellular machinery. These intimate interactions enable the parasites to gain unimpeded access to cytosolic resources critical for development while evading host immune responses. This review consolidates the latest advancements in understanding the molecular machinery driving these transkingdom contacts and their functional roles. Further investigation into these processes promises to revolutionize our understanding of organelle communication, with profound implications for identifying new therapeutic targets and strategies.

() promastigotes secrete exosomes that are crucial in host-pathogen interactions and intercellular communication by carrying parasite-specific molecules. Although the composition of cargos in exosomes is known, the effects of the unique metabolic repertoire on immunometabolism rewiring of macrophage polarization are poorly understood. Interestingly, we found the enrichment of polyamines (PAs) such as spermidine and putrescine in the -exosomes. Herein, we investigate the critical polycationic molecules and their crucial role in parasite survival. Our study shows that PA inhibition or depletion significantly impairs parasite growth and fitness, particularly in drug-resistant strains. Furthermore, we aimed to elucidate the impact of PAs-enriched -exosomes on host macrophages. The data demonstrated that macrophages efficiently internalized these exosomes, leading to heightened phagocytic activity and infectivity. In addition, internalized -exosomes induced M2 macrophage polarization characterized by elevated Arginase-1 expression and activity. The increased expression of the solute carrier gene (SLC3A2) and elevated intracellular spermidine levels suggest that -exosomes contribute to the host PAs pool and create an anti-inflammatory milieu. These findings highlight the essential role of PAs-enriched -exosomes in parasite survival and establishing a pro-parasitic environment in the host macrophage.

is the most ancient human tuberculosis pathogen and has been the leading cause of death from bacterial infectious diseases throughout human history. According to the World Health Organization Global Tuberculosis Report, in 2022, 7.5 million new tuberculosis cases were identified, marking the highest number of cases since the World Health Organization initiated its worldwide tuberculosis surveillance program in 1995. The 2019 peak was 7.1 million cases, with 5.8 million cases in 2020 and 6.4 million in 2021. The increase in 2022, which may be attributed to the COVID-19 pandemic complicating tuberculosis case tracing, has raised concerns. To better understand the regulation spectrum of under hypoxia, we performed a transcriptome analysis of mutant and wild-type strains using Illumina Agilent 5300 sequencing. The study identified 6898 differentially expressed genes, which were annotated with NCBI nonredundant protein sequences, a manually annotated and reviewed protein sequence database, Pfam, Clusters of Orthologous Groups of Proteins, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes. Several mycobacteria transcriptional regulators, virulence genes, membrane transporters, and cell wall biosynthesis genes were annotated. These data serve as a valuable resource for future investigations and may offer insight into the development of drugs to combat infection.

The rise of antibiotic-resistant Gram-positive pathogens, particularly methicillin-resistant (MRSA), presents a significant challenge in clinical settings. There is a critical need for new antibacterial agents to combat these resistant strains. Our study reveals that the uricosuric drug Benzbromarone (Benz) exhibits potent antibacterial activity against Gram-positive pathogens, with minimum inhibitory concentrations (MICs) ranging from 8 to 32 μg/mL and minimum bactericidal concentrations (MBCs) ranging from 32 to 128 μg/mL against clinical isolates of , , , and . Furthermore, Benz significantly inhibits biofilm formation at subinhibitory concentrations and eradicates mature biofilms at higher concentrations. Benz also suppresses the hemolytic activity of , indicating its potential to reduce virulence. Proteomic and induced resistance analyses indicate that Benz inhibits protein synthesis and turnover. Additionally, Benz induces membrane depolarization and increases membrane permeability, likely by targeting the membrane phospholipid phosphatidylethanolamine (PE). In the mouse wound infection model, Benz promotes wound healing and significantly reduces bacterial load. These findings suggest that Benz is a promising candidate for developing new antibacterial therapies against Gram-positive bacterial infections.

The urgent need for rapidly acting compounds in the development of antimalarial drugs underscores the significance of such compounds in overcoming resistance issues and improving patient adherence to antimalarial treatments. The present study introduces a high-throughput screening (HTS) approach using 1536-well plates, employing lactate dehydrogenase (PfLDH) combined with nitroreductase (NTR) and fluorescent probes to evaluate inhibition of the growth of the asexual blood stage of malaria parasites. This method was adapted to efficiently assess the speed of action profiling (SAP) in a 384-well plate format, streamlining the traditionally time-consuming screening process. By successfully screening numerous compounds, this approach identified fast-killing hits early in the screening process, addressing challenges associated with artemisinin-based combination therapies. The high-throughput SAP method is expected to be of value in continuously monitoring fast-killing properties during structure-activity relationship studies, expediting the identification and development of novel, rapidly acting antimalarial drugs within phenotypic drug discovery campaigns.

Polymicrobial wound infections caused by Gram-negative bacteria and associated inflammation are challenging to manage, as many antibiotics do not work against these infections. Utilizing adjuvants to repurpose the existing antibiotics for mitigating microbial infections presents an alternative therapeutic strategy. We designed and developed a niacin-cholic acid-peptide conjugate () to rejuvenate the therapeutic efficacy of macrolide antibiotics against Gram-negative pathogens. We conjugated niacin with anti-inflammatory properties at the carboxyl terminal of the cholic acid and dipeptide (glycine-valine) at the three hydroxyl terminals of cholic acid to obtain the amphiphile . Our findings demonstrated that amphiphile serves as a microbial membrane disruptor that facilitates the entry of erythromycin (ERY) in bacterial cells. The combination of amphiphile and ERY is bactericidal and can effectively eliminate monomicrobial and polymicrobial Gram-negative bacterial biofilms. We further demonstrated the antibacterial effectiveness of combining and ERY against monomicrobial and polymicrobial wound infections. Together, these findings indicate that amphiphile revitalizes the remedial efficacy of ERY against Gram-negative bacteria.

Fascioliasis, a zoonotic disease caused by liver flukes of the genus , poses significant health threats to both humans and livestock. While some infections remain asymptomatic, others can lead to fatal outcomes, particularly during the acute phase characterized by the migration of immature parasites causing severe liver damage. Through the combination of data acquired via high-spatial-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) and nanohydrophilic interaction chromatography tandem mass spectrometry, we investigated glycosphingolipids (GSLs) in both adult and immature parasite stages as well as the host liver and bile duct to unravel the intricacies of the host-pathogen interplay and associated pathology. Several GSLs showed characteristic distribution patterns within the parasite depending on the fatty acid composition of their ceramides, notably including GSLs carrying very long-chain fatty acids. Additionally, GSL compositions within the tegument of immature versus adult parasites varied, suggestive of tissue remodeling upon maturation. AP-SMALDI MSI further enabled the identification of GSLs potentially involved in interactions between the host and immature parasites. Moreover, our experiments unveiled alterations in other lipid classes during infection, providing a broader understanding of lipidomic changes associated with the disease. Collectively, our findings contribute to a deeper comprehension of the molecular intricacies underlying fascioliasis, with a specific focus on GSLs.

is a leading bacterial pathogen that causes persistent infections. One major reason that antibiotics fail to clear such infections is the presence of a dormant subpopulation called persister cells. To eradicate persister cells, it is important to change drug development from traditional strategies that focus on growth inhibition to the search for new leads that can kill dormant cells. In this study, we demonstrate that eravacycline can effectively accumulate in persister cells, leading to strong killing during wakeup, including persister cells in both planktonic cultures and biofilms of the wild-type strain and its mucoid mutant. The effects of eravacycline on persister control were further validated using a lung infection model in mice. Collectively, these results demonstrate the possibility to control persister cells of bacterial pathogens by targeting dormancy.

Coinfection of (Mtb) and human immunodeficiency virus-1 (HIV) is a significant public health concern. Treatment is challenging due to prolonged duration of therapy and drug interactions between antiretroviral therapy (ART) and anti-TB drugs. Noniron gallium -tetraphenyl porphyrin (GaTP), a heme mimetic, has shown broad antimicrobial activity. Here, we investigated the efficacy of nanoparticle encapsulating GaTP (GaNP) for the treatment of HIV and Mtb coinfection or single infection in granuloma structures. GaNP significantly reduced viable Mtb within primary human granuloma structures infected with Mtb H37Rv-lux and significantly reduced levels of HIV in CD4+ T cells infected with the virus axenically. Similarly, GaNP exhibited significant antimicrobial activity against HIV/Mtb-coinfected granuloma structures created , which contain the primary immune cells seen in human TB granulomas, including CD4+ T cells and macrophages, as assessed by a luciferase assay for Mtb and p24 ELISA for HIV detection. Furthermore, mechanistic studies revealed that GaTP increases the level of reactive oxygen species and inhibits catalase in Mtb. A significant increase in Mtb nitrate reductase activity was also observed when Mtb was incubated with GaTP and sodium nitrate. Overall, increased oxidative stress and nitrite levels induced by GaTP are consistent with the possibility that GaTP inhibits Mtb aerobic respiration, which leads to incomplete O reduction and a shift to respiration using exogenous NO. These cumulative data continue to support the potential for developing the noniron heme analog GaTP and its nanoparticle GaNP as new therapeutic approaches for the treatment of HIV/Mtb coinfection.

The Zika virus (ZIKV) has garnered significant public attention, particularly following the outbreak in Brazil, due to its potential to cause severe damage to the central nervous system and its ability to cross the placental barrier, resulting in microcephaly in infants. Despite the urgency, there remains a lack of targeted therapies or vaccines for the prevention or treatment of ZIKV infection and its related diseases. Fangchinoline (FAN), an alkaloid derived from traditional Chinese medicinal herbs, has a range of biological activities. In this study, we employed both and infection models to demonstrate the efficacy of FAN in inhibiting ZIKV. Our findings indicate that FAN effectively suppresses the replication of ZIKV viral RNA and protein, thereby validating its anti-ZIKV capabilities in living organisms. Further analysis through dosing time assays and infectious inhibition assays revealed that FAN exerts its antiviral effects by impeding the early stages of infection, specifically by inhibiting the internalization of ZIKV. These results underscore the potential of FAN as a candidate for anti-ZIKV drug development and offer novel insights into drug design strategies that target the virus's internalization process.

are foodborne parasites that opportunistically parasitize humans, leading to acute abdominal symptoms and allergies. Besides gastroscopy, no other diagnostic technique is available. Consequently, it is necessary to identify specific biomarkers and then develop molecular techniques for diagnosing infection. In the present study, we used immunoproteomic and immunoinformatic approaches to identify sensitive antigens for diagnosing infection. A total of three proteins, including Ani609 (VDK51609), Ani941 (VDK75941), and AniS13, were identified based on immunoinformatic results. Then, the indirect ELISA method was developed based on the recombinant proteins, showing a similar diagnostic capability to that of parasitic soluble proteins. Next, a luciferase immunoprecipitation assay (LIPS) was further developed upon the fusion of the proteins and luciferase. The LIPS method indicated that infection could be detected in rats as early as 1 week post infection, especially for Ani941. Overall, we identified the novel antigens using immunoproteomic and immunoinformatic approaches and then developed a sensitive method for diagnosing infection, particularly for the early stage.

Worldwide, bacterial antibiotic resistance continues to outpace the level of drug development. One way to counteract this threat to society is to identify novel ways to rapidly screen and identify drug candidates in living cells. Developing fluorescent antibiotics that can enter microorganisms and be displaced by potential antimicrobial compounds is an important but challenging endeavor due to the difficulty in entering bacterial cells. We developed a cell-based assay using a fluorescent aminoglycoside molecule that allows for the rapid and direct characterization of aminoglycoside binding in a population of bacterial cells. The assay involves the accumulation and competitive displacement of a fluorescent aminoglycoside binding probe in as a Gram-negative bacterial model. The assay was optimized for high signal-to-background ratios, ease of performance for reliable outcomes, and amenability to high-throughput screening. We demonstrate that the fluorescent binding probe shows a decrease in fluorescence with cellular uptake, consistent with RNA binding, and also shows a subsequent increase upon the addition of the positive control neomycin. Fluorescence intensity increase with aminoglycosides was indicative of their relative binding affinities for A-site rRNA, with neomycin having the highest affinity, followed by paromomycin, tobramycin, sisomicin, and netilmicin. Intermediate fluorescence was found with plazomicin, neamine, apramycin, ribostamicin, gentamicin, and amikacin. Weak fluorescence was observed with kanamycin, hygromycin, streptomycin, and spectinomycin. A high degree of sensitivity was observed with aminoglycosides known to be strong binders for the 16S rRNA A-site compared with antibiotics that target other biosynthetic pathways. The quality of the optimized assay was excellent for planktonic cells, with an average ' factor value of 0.80. In contrast to planktonic cells, established biofilms yielded an average ' factor of 0.61. The high sensitivity of this cell-based assay in a physiological context demonstrates significant potential for identifying potent new ribosomal binding antibiotics.

Antimicrobial resistance (AMR) poses a serious threat to global health. The rapid emergence of resistance contrasts with the slow pace of antimicrobial development, emphasizing the urgent need for innovative drug discovery approaches. This study addresses a critical bottleneck in early drug development by introducing integral solvent-induced protein precipitation (iSPP) to rapidly assess the target-engagement of lead compounds in extracts of pathogenic microorganisms under close-to-physiological conditions. iSPP measures the change in protein stability against solvent-induced precipitation in the presence of ligands. The iSPP method for bacteria builds upon established SPP procedures and features optimized denaturation gradients and minimized sample input amounts. The effectiveness of the iSPP workflow was initially demonstrated through a multidrug target-engagement study. Using quantitative mass spectrometry (LC-MS/MS), we successfully identified known drug targets of seven different antibiotics in cell extracts of four AMR-related pathogens: the three Gram-negative bacteria , , and the Gram-positive bacterium . The iSPP method was ultimately applied to demonstrate target-engagement of compounds derived from target-based drug discovery. We employed five small molecules targeting three enzymes in the 2--methyl-d-erythritol 4-phosphate (MEP) pathway─a promising focus for anti-infective drug development. The study showcases iSPP adaptability and efficiency in identifying anti-infective drug targets, advancing early-stage drug discovery against AMR.

malaria parasites retain an essential mitochondrional electron transport chain (ETC) that is critical for growth within humans and mosquitoes and is a key antimalarial drug target. ETC function requires cytochromes and , which are unusual among heme proteins due to their covalent binding to heme via conserved CXXCH sequence motifs. Heme attachment to these proteins in most eukaryotes requires the mitochondrial enzyme holocytochrome synthase (HCCS) that binds heme and the apo cytochrome to facilitate the biogenesis of the mature cytochrome or . Although humans encode a single bifunctional HCCS that attaches heme to both proteins, parasites are like yeast and encode two separate HCCS homologues thought to be specific for heme attachment to cyt (HCCS) or cyt (HCCS). To test the function and specificity of HCCS and HCCS, we used CRISPR/Cas9 to tag both genes for conditional expression. HCCS knockdown selectively impaired cyt biogenesis and caused lethal ETC dysfunction that was not reversed by the overexpression of HCCS. Knockdown of HCCS caused a more modest growth defect but strongly sensitized parasites to mitochondrial depolarization by proguanil, revealing key defects in ETC function. These results and prior heterologous studies in of cyt hemylation by HCCS and HCCS strongly suggest that both homologues are essential for mitochondrial ETC function and have distinct specificities for the biogenesis of cyt and , respectively, in parasites. This study lays a foundation to develop novel strategies to selectively block ETC function in malaria parasites.