Colorectal cancer, which originates in the epithelial cells of the colon or rectum, is closely associated with dysbiosis of the gut microbiota. Increasing evidence has shown that plays a significant role in colorectal cancer progression by activating inflammatory responses, modulating the tumor microenvironment, and promoting tumor cell proliferation. Antimicrobial peptides targeting have the potential to serve as more effective and less toxic therapeutic agents compared to chemotherapy drugs. In this study, we systematically evaluated the antibacterial activity of Trp-containing peptides, including natural peptides isolated from the skin secretions of the Chinese brown frog () and their derivatives, which exhibit potent antibacterial activity against with minimal cytotoxicity. Mechanistic investigations using membrane permeability assays and membrane potential-sensitive dyes indicated that Trp-containing peptides exert their antimicrobial effects by disrupting the bacterial membrane structure, increasing membrane permeability, and interfering with membrane potential. In a colorectal cancer mouse model infected with , treatment with Trp-containing peptides significantly alleviated tumor-related symptoms, reduced colonic inflammatory cytokine levels, and alleviated colonic tissue damage, as confirmed by histopathological analysis. Importantly, no apparent toxicity or adverse effects were observed during the treatment. These findings indicate that Trp-containing peptides, as lead compounds, not only exhibit potent antibacterial activity but also attenuate associated colorectal cancer progression, providing critical evidence to support the development of innovative therapeutic strategies with combined antimicrobial and antitumor properties.

is a major etiologic agent for infectious bovine keratoconjunctivitis (IBK), commonly known as bovine pink eye. IBK has been a major economic burden to the cattle and dairy industries due to its economic and welfare impacts on affected cattle herds. Antimicrobial treatment of acute IBK infections is often challenging. Vaccine formulations widely used in industry have poor efficacy for the prevention of IBK. Capsular polysaccharides of some bacterial pathogens are important epidemiological markers and are successfully used in vaccines for humans. Currently, there are limited data demonstrating the presence of capsular polysaccharides in . In this study, we show by genomic analysis that a broad selection of strains obtained from the eyes of cattle harbor a gene cluster for expressing capsular polysaccharides. The isolates potentially express either a chondroitin-like polysaccharide or an α(2-8) polysialic acid. We isolated a polysaccharide from cultures of a well-studied model strain for IBK, the Epp63 strain, structurally identical to capsule α(2-8) polysialic acid of the human pathogens K1 and Group B. The gene cluster in Epp63 encodes a polysialyltransferase similar to other bacterial polysialyltransferases. Other strains analyzed in this study possess a gene homologous to that of bacterial chondroitin synthase. We isolated a capsular polysaccharide from genotypes 1 and 2 that has the repeat unit identical to nonsulfated chondroitin. These findings provide a tool for the study of IBK pathogenesis that could lead to approaches for better control of the disease.

Plasmepsin V (PMV), an essential aspartyl protease, plays a critical role during the asexual blood stage of infection of by enabling the export of parasite proteins into the host red blood cell. This export is vital for parasite survival and pathogenesis, making PMV an attractive target for antimalarial drug development. Peptidomimetic inhibitors designed to mimic the natural substrate of PMV have demonstrated potent parasite-killing activity by blocking protein export. While these compounds have been instrumental in validating PMV as a antimalarial target, inconsistencies between their biochemical potency and cellular activity have raised questions regarding their precise mechanism of action. In this study, we employed chemoproteomic approaches, including solvent-induced protein precipitation and intact-cell thermal profiling, to demonstrate PMV target engagement by the peptidomimetics. To further support these findings, we generated parasite lines exhibiting reduced sensitivity to peptidomimetics. Through whole-genome sequencing of these parasite lines, a single nucleotide variant within the gene was revealed. This mutation was later validated using reverse genetics, confirming its role in mediating resistance. Together, these data provide strong evidence that the peptidomimetics exert their antimalarial activity by directly targeting PMV. These findings further support the potential of PMV as a validated and promising target for future antimalarial drug development.

poses a global threat to livestock health and public health security, necessitating a novel antibiotic. Pleuromutilin, a natural antibiotic, has served as a promising foundation for developing new antibacterial agents through structural modification. This study aims to evaluate the antibacterial potential, safety, and pharmacokinetic profile of a novel pleuromutilin derivative 2-3 (22-[2-(L-prolylamino)phenylsulfanyl]-22-deoxypleuromutilin). The compound 2-3 exhibited potent antibacterial activity (MIC = 0.25 μg/mL), concentration-dependent bactericidal effects, and prolonged post-antibiotic effects (PAEs). Safety assessments revealed low cytotoxicity (CC = 62.63 μg/mL) and no observable hemolytic activity. In vitro metabolic studies indicated species-dependent clearance, primarily mediated by CYP3A4. Pharmacokinetic in rats showed rapid absorption and elimination, with oral and intramuscular bioavailability of 16.03% ± 8.82% and 53.36% ± 12.27%, respectively. Notably, 2-3 demonstrated superior efficacy over tiamulin in a neutropenic murine thigh infection model. Molecular docking revealed a stronger binding free energy between 2-3 and the 50S ribosomal subunit compared to tiamulin. Collectively, these results highlight 2-3 as a promising clinical candidate against infections, characterized by enhanced efficacy and a favorable safety profile.

Chagas disease (CD), caused by , has been one of the leading causes of cardiac death in Latin America. Its pathogenesis and progression are still poorly understood. Thus, we performed an untargeted metabolomics analysis to understand the metabolic changes involved in the final acute phase of CD. Male mice's chagasic hearts (60 days postinfection) were compared to healthy tissues. Two hundred and fifty-one significant metabolites or chemical classes were annotated. Disturbances in energy metabolism and dysregulation of amino acids were observed. Pathway analyses indicated increased inflammatory activity in infected individuals, as observed by eicosanoid (prostaglandin and thromboxane) changes. The accumulation of some sphingomyelins, correlated with myocarditis, suggests heart tissue damage from the infection. The metabolic changes observed contribute to understanding disease progression and the cardiac effects caused by the parasite, bringing new insights into the discovery and development of new therapies.

, an intracellular protozoan parasite, resides within parasitophorous vacuoles in host macrophages and relies on host-pathway manipulation for survival. Here, we uncover a novel role of the surface metalloprotease GP63 in stabilizing the parasitophorous vacuoles through targeted subversion of host vesicular trafficking and apoptosis. We demonstrate that GP63 is essential for the selective recruitment of the Golgi-associated adaptor protein PIST to the parasitophorous vacuoles, a process that is impaired in GP63-deficient (LmGP63) parasites. GP63 facilitates PIST-Golgin160 complex formation by suppressing caspase-3 activation, preventing Golgin160 cleavage. Caspase inhibition via Z-VAD-FMK further enhances this complex's recruitment. Moreover, GP63 selectively modulates autophagy by promoting PIST-Beclin1 colocalization while excluding LC3 from the parasitophorous vacuoles. These findings identify GP63 as a central effector that orchestrates host vesicular and apoptotic pathways to maintain parasitophorous vacuoles integrity and promote chronic infection, offering insights into potential therapeutic targets against Leishmaniasis.

Methicillin-resistant (MRSA) ST398 carries two hyaluronidase genes, and its homologue , the latter located on the genomic island νSaβ. However, the prevalence of and its contribution to virulence remain unclear. Here, we report that the gene is present in 18.3% (4707/25,752) of in the NCBI database, with ST398 being the most prevalent sequence type (30.9%, 1457/4707). In ST398, the gene is flanked by IS and IS, with >99.0% nucleotide identity across strains, suggesting horizontal acquisition. In a mouse skin infection model, a wild-type ST398 MRSA strain carrying both and formed significantly larger abscesses than isogenic mutants lacking one or both hyaluronidase genes. Wild-type infection led to a higher bacterial load and sustained induction of chemokines (CCL5, CXCL1, CCL4) and pro-inflammatory cytokines (IL-1β, IL-6, IL-33), resulting in prolonged neutrophil recruitment and severe inflammation. Consistently, and enhanced the survival of MRSA ST398 inside RAW 264.7 macrophages and neutrophils. , a double knockout strain (Δ-Δ) grew more slowly with hyaluronic acid (HA) as the sole carbon source, accompanied by intracellular accumulation of specific amino acids (proline, valine, threonine, and phenylalanine) and downregulation of amino acid biosynthesis pathways. Moreover, RAW 264.7 macrophages infected with Δ-Δ showed a marked upregulation of the oxidative phosphorylation (OXPHOS) pathway compared to uninfected controls, suggesting an enhanced cellular metabolic and inflammatory response that could improve bacterial clearance. Our findings highlight the functionally redundant roles of and in MRSA ST398 pathogenesis, suggesting that these hyaluronidases are potential targets for antistaphylococcal therapy.

(), the causative agent of human tuberculosis (TB), employs its de novo histidine (His) biosynthesis to escape host-inflicted His starvation. This makes the enzymes involved in this biosynthetic pathway promising anti-TB drug targets. In this study, employing the high-resolution crystal structure of imidazole glycerol phosphate dehydratase (IGPD) of the His pathway, three triazole scaffold molecules were identified as potential inhibitors of this enzyme. These high-resolution crystal structures of the enzyme-inhibitor complexes elucidated the key interactions responsible for their binding specificity and affinity. We also studied the interactions of these inhibitors with the enzyme at the atomic level and tested their cytotoxicity and efficacy in and models. Our findings revealed that the most prominent inhibitor, SF2, was safe in mice and effectively inhibited the growth of both free as well as in macrophage-internalized wild-type and drug-resistant clinical isolates. Notably, SF2 also showed a marginal reduction in the bacterial load in organs of mice infected with . Collectively, these findings advocate the chemical inhibition of IGPD of the His pathway as a novel anti- therapeutic strategy.

Cestodes (class Cestoda) include zoonotic parasites such as spp. and spp., which cause significant morbidity and mortality in endemic regions, particularly among pastoral and rural populations in low-, upper-, and middle-income countries. Their developmental plasticity reflects finely tuned regulatory mechanisms controlling gene expression throughout complex life cycles and infection stages. Despite expanding genomic resources, functional postgenomic studies remain scarce. MicroRNAs (miRNAs) are critical regulators of gene expression, influencing diverse developmental and physiological processes. Among them, miR-71-5p is highly expressed in cestodes, absent in vertebrates, and predicted to regulate essential parasite genes. Here, we employed a chemically modified antisense oligonucleotide to assess the impact of miR-71-5p knockdown during infection. To the best of our knowledge, this represents the first report of miRNA knockdown in a cestode infection model. Treated mice exhibited a 67% (3-fold) reduction in parasitic mass compared with controls, suggesting that miR-71-5p is essential for infection establishment and progression. Toxicity analyses in uninfected mice revealed no adverse effects. Whole-mount hybridization showed broad miR-71-5p expression across tissues, including germinative cells, suggesting a pleiotropic role. These findings advance the understanding of miRNA-mediated regulation in cestodes and highlight these small RNAs as promising therapeutic targets for neglected tropical diseases (NTDs) prioritized by the World Health Organization (WHO).

The FF-ATP synthase is essential to the aerobe . Its F-domain utilizes the proton motive force to rotate the turbine (-ring) inside the stator ( subunit), which generates a torque that is translated to the catalytic F-domain for adenosine 5'-triphosphate (ATP) synthesis. Here, we investigated key features of the F-domain, including the proton intake channel, proton donor and acceptor residues, an unique subunit helix, and the proton exit pathway. By employing a heterologous system, we generated mutants and studied their growth kinetic properties in minimal media, as well as the ATP synthesis activity of their inverted-membrane vesicles. The findings highlight the front entry as the main proton uptake pathway and the key residues involved in proton translocation. Molecular dynamics (MD) simulations confirm the role of these charged residues, which interact with water molecules to facilitate a water-mediated proton transfer in a Grotthuss-like mechanism. Similarly, the exit channel with R224 of subunit playing a central role is described. Importantly, the sequential flow of proton intake, turbine rotation, and proton release are modulated by the unique subunit helix, which functions like a molecular ratchet to facilitate effective proton transfer for the final formation of ATP. The importance in function, difference in amino acid content, and uniqueness in regulation by its specific molecular ratchet make the proton pathway an attractive inhibitor target, where a cork-like molecule could prevent proton intake and/or release with the consequence of ATP synthesis and cell growth inhibition.

Resistance to artemisinin-based combination therapies (ACTs) is steadily increasing in malaria-endemic countries, and new medicines to treat this disease are urgently needed. Drug discovery efforts are hindered by species differences in drug metabolism as new chemical entities must survive metabolism by diverse enzymes across multiple species, enabling cures in preclinical disease models before progression to the clinic. Here, we show how the use of a mouse line extensively genetically humanized for enzymes of the cytochrome P450 superfamily and their transcriptional regulators, the "8HUM" line, can circumvent this issue and improve the translational accuracy of data generated. Engraftment of human erythrocytes into 8HUM/Rag2, an immunocompromised version of the 8HUM line lacking mature T and B cells, was insufficient to permit infection with , and depletion of natural killer cells by antibody treatment did not alter this outcome. However, infection of 8HUM with permitted assessment of drug efficacy against this species. Approved antimalarials were generally more metabolically stable in 8HUM than in wild-type mice. Major species differences between humans and mice in routes of metabolic elimination for quinine derivatives were removed with 8HUM. Therefore, the 8HUM model described here will be of value early in the critical path for antimalarial drug discovery, improving alignment of drug metabolism with the clinical situation while bypassing mouse-specific issues of metabolism to facilitate proof-of-concept in vivo demonstration of efficacy, a key requirement for validation of new drug targets and chemical series.

is an early colonizer of the oral microbiome and contributes positively to oral health. While this species has been found to produce hydrogen peroxide by expression, the relationship of this expression to the competence regulon has not yet been explored. To this end, this study sought to investigate the connection of the competence regulon quorum sensing (QS) circuitry with downstream proliferative phenotypic expression resulting from competence-stimulating peptide (CSP) exposure, with specific attention to peroxide formation. Following confirmation of the native CSP, RNA-seq was completed to gain insights into transcriptomic variations resulting from CSP incubation. Later, structure-activity relationship (SAR) analyses of the native CSP were completed. The results revealed residues integral to CSP:ComD binding and activation, while indicating which residues were considered dispensable to this process. Phenotypic assessment revealed that peroxide formation was modulated via the competence regulon. Finally, interspecies competition assays were carried out to understand the interactions between and , with demonstrating a profound capability of antagonizing growth and proliferation. Our results support that this antagonism is mainly attributed to hydrogen peroxide production by . This finding suggests that may be exploited for its beneficial proliferative phenotypes downstream of the competence regulon.

big defensins (-BigDefs) are a family of two-domain antimicrobial peptides with broad antibacterial activity. The C-terminal domain of -BigDefs harbors a β-defensin-like structure, whereas the ancestral N-terminal domain adopts a globular structure. Here, we developed molecular tools to track the fine interactions of these two domains with and to gain insight into BigDef1 mechanisms of action. By using super-resolution microscopy and mutants with specific deletions of cell wall components, we found that teichoic acids (TAs) play a key role in the BigDef1 interaction with . A Δ mutant lacking cell wall teichoic acids (WTAs) exhibited increased resistance to BigDef1. Consistently, the binding of BigDef1 to cell wall was significantly reduced in the Δ mutant. In contrast, a Δ mutant unable to transfer d-alanine onto lipoteichoic acid (LTA) showed increased susceptibility to BigDef1 and lysed rapidly in contact with the peptide. BigDef1 bound to cell wall. In addition, competitive binding with exogenously added LTA was sufficient to impair BigDef1 antimicrobial activity against . These data suggest that TAs are conserved molecular motifs recognized by BigDef1. Finally, we found that BigDef1 interaction with was mediated by its N-terminal domain, which enables the C-terminal β-defensin-like domain to interact with the bacterial cell wall. Altogether, our results identify TAs as important targets for BigDef1. This interaction appears to be mediated by the ancestral N-terminal domain characteristic of this peptide family.

Polymyxins are considered last-resort antibiotics for multidrug-resistant Gram-negative bacteria. However, their clinical utility is limited by toxicities, particularly nephrotoxicity and neurotoxicity, as well as the emergence of resistance. This review addresses these challenges and evaluates strategic interventions aimed at enhancing the efficacy of polymyxins while mitigating their adverse effects. Combination therapies have emerged as a cornerstone strategy. These therapies can be categorized into five frameworks: structural barrier disruption, bioenergetic flux modulation, metabolic homeostasis disruption, resistance neutralization, and virulence disarming. In addition to synergistic agents, complementary strategies such as detoxifying adjuvants and advanced delivery systems have been systematically integrated to overcome the intrinsic limitations of polymyxins. Collectively, these multifaceted strategies enhance the antibacterial activity of polymyxins against Gram-negative bacteria, while simultaneously reducing effective doses, minimizing toxicity, and mitigating the development of resistance. These innovations represent a pivotal advance in revitalizing polymyxin therapy in the era of multidrug resistance.

Being a persistent and deadly infection, tuberculosis (TB) caused by remains a global health challenge. Despite having a well-established 4-drug combination therapy for drug-sensitive TB, the emergence of drug-resistant TB has rendered the treatment less effective. Although fluoroquinolones (FQs) are among the prominent drugs in the second-line treatment for multidrug-resistant tuberculosis (MDR-TB), FQ resistance has readily emerged in cases of extensively drug-resistant tuberculosis (XDR-TB). Other than the mutations in DNA gyrase, a universally conserved bacterial enzyme targeted by FQs, several mechanisms contribute to the emergence of FQ resistance. Recently, post-translational modification of DNA gyrase is implicated as one of the mechanisms for FQ resistance. Here, we describe succinylation of GyrB by a promiscuous acetyltransferase, Eis of , as a new mechanism contributing to FQ resistance in mycobacteria. Lysine succinylation of GyrB results in a reduced interaction of FQs with the enzyme, thereby decreasing the DNA cleavage by DNA gyrase in the presence of FQs. Accordingly, Eis overexpressing mycobacterial strains exhibit increased minimum inhibitory concentration (MIC) to FQs. Thus, succinylation of DNA gyrase is yet another resistance mechanism against the FQs.

Bacterial strains are distinguished by surface glycans composed of defined sugar sequences that include "rare" monosaccharides, which are absent in human glycans and help to mediate host-microbe interactions. One of the most prevalent rare sugars is l-Rhamnose (l-Rha), and human sera are generally enriched in anti-l-Rha antibodies; however, the source of l-Rha antigens is unknown. Here, we synthesize a surface glycan l-Rha--acetyl glucosamine disaccharide sequence, which is found across many bacterial species, to evaluate binding motifs of human anti-glycan antibodies in clinical and commercial human sera. We find that sera are enriched in IgG antibodies that react with this disaccharide probe. Through capture of bound antibodies and analysis with surface glycan sequences from different strains, we observe that bound human antibodies appear to recognize free or branched, but not internal, l-Rha motifs. Overall, this work details the isolation of naturally occurring anti-l-Rha human antibodies and promotes an understanding of their carbohydrate recognition epitopes.

Bacterial and human cells produce extracellular vesicles (EVs) in response to diverse stimuli, e.g., toxins, oxidative stress, nutrient depletion, or high cell density. Here, we describe a cocultivation platform that allows recovery of mixed extracellular vesicles (mix-EVs) produced simultaneously by both cell types. We investigated interactions between Gram-positive and Gram-negative bacteria (, , , and ) and human peripheral blood mononuclear cells (PBMCs). The production of the mix-EVs population decreased with higher bacterial concentrations. Exposing PBMCs to mix-EVs repressed the general transcriptomic signature, in contrast with a significant upregulation generated by single bacterial-EVs. However, mix-EVs-derived IL-1β upregulation was confirmed at the protein level. Inhibition experiments showed that IL-1β production involved TLR2 and TLR4 signaling, acting through IRAK-1 and TRAF6 related pathways. This approach provides a new platform for the study of EVs at the pathogen-host interface and presents mechanistic insights into the effect of EVs on an infected host.

Bacterial glycans are complex and often presented on the surface of the cell as a level of protection. These glycans offer an opportunity to screen for new antibiotic targets and immunological markers. Here recent developments in the field of glycan arrays are presented as opportunities to advance therapies for human health.