Search Results (121)

Variants in CLCN3 and CLCN4, encoding the neuronal endosomal Cl⁻/H⁺ antiporters ClC-3 and ClC-4, are linked to neurodevelopmental disorders with broad phenotypic variability. Over sixty CLCN4 variants have been functionally characterized, showing gain- or loss-of-function (GoF or LoF) effects. While ClC-3 can function as a homodimer, ClC-4 depends on heterodimerization with ClC-3 for efficient endosomal trafficking. CLCN4, located on the X chromosome, exhibits diverse pathogenic outcomes: complete LoF variants often cause non-syndromic presentations in hemizygous males and are asymptomatic in heterozygous females, whereas certain missense variants with partial or complete LoF produce severe syndromic phenotypes in both sexes. Here, we demonstrate dominant effects of three CLCN4 variants within ClC-3/ClC-4 heterodimers using two-electrode voltage-clamp recordings in Xenopus laevis oocytes and whole-cell patch-clamp recordings in mammalian cells co-expressing both proteins via a bicistronic IRES construct. Our findings provide the first evidence of dominant-negative CLCN4 effects within ClC-3/ClC-4 complexes and establish a platform for functional analysis of additional disease-associated variants. Full article

Functionally similar to a plant vacuole or a mammalian lysosome, the fungal vacuole plays a vital role in many cellular processes. Most studies of the vacuole have been performed in the nonpathogenic yeast Saccharomyces cerevisiae; however, the vacuole in pathogenic fungi has recently been implicated in host invasion in both plants and mammals, highlighting an important role for the vacuole in pathogenesis. Here, we report that deletion of C. neoformans vacuolar protein 8 (VAC8) results in fragmented vacuole morphology, impairment of vacuolar fusion, and inability to form titan cells. Additionally, absence of Vac8 results in defective growth at high temperature and in the presence of caffeine, suggesting a defect in cell wall signaling. Interestingly, despite aberrant vacuole morphology, vac8Δ is slightly more resistant to fluconazole treatment, and displays increased resistance to hydrogen peroxide, suggesting the irregular vacuole morphology does not impair vacuolar function. Like S. cerevisiae Vac8, C. neoformans Vac8 is comprised of armadillo repeat regions which form alpha helices that fold to form a superhelix, allowing for increased protein–protein interaction. Many of the known binding partners of S. cerevisiae Vac8 are not present in the C. neoformans genome, suggesting novel functions for Vac8 in this fungus. Notably, deletion of VAC8 affected some virulence traits, providing support for targeting the fungal vacuole as a potential therapeutic intervention. Full article

Glycosylphosphatidylinositols (GPIs) are complex glycolipids that function as membrane anchors for a wide array of eukaryotic proteins, collectively referred to as GPI-anchored proteins (GPI-APs). These structures are critical for various cellular processes including signal transduction, host–pathogen interactions, and immune evasion. While GPI-APs have been extensively studied, increasing attention is being paid to non-protein-linked GPI, called free GPIs, which have been identified in both protozoan parasites and mammalian cells. In protozoa such as Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondii, Plasmodium falciparum, and Leishmania spp., free GPIs play roles in virulence, immune modulation, and parasite survival. In mammals, free GPIs have been detected in several tissues and pathogenic conditions of paroxysmal nocturnal hemoglobinuria caused by PIGT mutation and rare blood group phenotypes. This review provides a comparative overview of the structure and biosynthesis of free GPIs and GPI-APs across species, highlighting unique adaptations in each. We also discuss the emerging physiological and pathological roles of free GPIs, proposing that these underexplored molecules may serve as important biomarkers and therapeutic targets. Understanding the diversity and function of free GPIs offers new insights into glycobiology and host–pathogen interactions. Full article

Post-translational modifications (PTMs) are critically important for protein structure and function, with glycosylation being one of the most common forms of PTM. The gastric pathogen Helicobacter pylori has a general glycosylation system, which performs complex glycosylation of lipopolysaccharide, flagella proteins, and outer membrane proteins (OMPs). One of the best-described OMPs of H. pylori is the blood group binding adhesin (BabA), which interacts with the Lewis histo-blood group antigen, Lewis b. The 3D structure for BabA has been determined, and the ligand specifically described. Although BabA is reported to be a glycoprotein, there are limited data examining the effects of glycosylation on the structure and function of this protein. This study examined the folding and thermostability of non-glycosylated recombinant BabA and used computational approaches to predict the effect of glycosylation on the protein, with a focus on its possible heterologous expression in mammalian cells. Three potential O-linked and three potential N-linked glycosylation sites were predicted. Furthermore, the effect of glycan shielding on the solvent-accessible surface area of BabA was examined. Molecular dynamics simulations highlighted local indicators, including root mean square fluctuation and the number of protein-glycan contacts that were affected by glycosylation. Taken together, the findings support a role of glycans in surface shielding and promoting local stabilization in specific areas of the BabA protein. This study helps to strengthen the understanding of the importance of glycosylation and the role it plays in the structure, function, and stability of H. pylori proteins. Full article

Piscine orthoreovirus (PRV) is a globally distributed viral pathogen that causes heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar) and affects other salmonids, yet no commercial vaccines are currently available. Major barriers to vaccine development include the inability to propagate PRV in cell lines and the low, variable immunogenicity of its proteins, particularly the outer capsid protein σ1, which mediates viral attachment. This protein is hypothesized to be immunologically relevant due to its homology with Mammalian orthoreoviruses. Recombinant σ1 expressed in conventional systems exhibits poor antibody recognition, whereas structural modifications such as lipidation or fusion with molecular chaperones improve epitope exposure. Formalin-inactivated vaccines have shown inconsistent protection, often failing to elicit robust innate or adaptive responses, especially under cohabitation challenge. In contrast, DNA vaccines encoding σ1 and the non-structural protein μNS have demonstrated partial efficacy, likely due to enhanced intracellular expression and antigen presentation. Nonetheless, the considerable variability observed in immune responses among individual fish and viral genotypes, together with suggestions that PRV may interfere with antiviral pathways, represent additional barriers to achieving consistent vaccine efficacy. This review summarizes the current status of PRV vaccine development and discusses future directions for rational design based on optimized antigens and intracellular delivery platforms. Full article

Animal venoms are rich in bioactive molecules with promising biotechnological potential. They comprise both protein and non-protein toxins. Among the protein toxins are enzymes, such as phospholipases A₂, proteases and L-amino acid oxidases (LAAOs). LAAOs exhibit antimicrobial, antiparasitic, antiviral, and anticancer effects, making them potential candidates for biotechnological applications. These activities are linked to their ability to catalyze oxidative reactions that convert L-amino acids into α-keto acids, releasing ammonia and hydrogen peroxide, which contribute to the immune response, pathogen elimination, and oxidative stress. However, in snakes of the Micrurus genus, LAAOs generally represent a small portion of the venom (up to ~7%), which limits their isolation and study. To overcome this, the present study aimed to produce Ml-LAAO, the enzyme from Micrurus lemniscatus, through heterologous expression in mammalian cells. The gene sequence was inferred from its primary structure and synthesized into the pSecTag2B vector for expression in HEK293T cells. After purification using a His Trap-HP column, the presence of recombinant Ml-LAAO (Ml-LAAOrec) was confirmed by Western blot and mass spectrometry, validating its identity. These results support successful recombinant expression of Ml-LAAO and highlight its potential for scalable production and future biotechnological applications. Full article

Pasteurella multocida (Pm) is a zoonotic pathogen that poses a significant threat to animal health and causes substantial economic losses, further aggravated by rising tetracycline resistance. To restore the efficacy of tetracyclines to Pm, we evaluated the synergistic antibacterial activity of doxycycline combined with metformin, an FDA-approved antidiabetic agent. Among several non-antibiotic adjuvant candidates, metformin exhibited the most potent in vitro synergy with doxycycline, especially against capsular serogroup A strain (PmA). The combination demonstrated minimal cytotoxicity and hemolysis in both mammalian and avian cells and effectively inhibited resistance development under doxycycline pressure. At 50 mg/kg each, the combination of metformin and doxycycline significantly reduced mortality in mice and ducks acutely infected with PmA (from 100% to 60%), decreased pulmonary bacterial burdens, and alleviated tissue inflammation and damage. Mechanistic validation confirmed that metformin enhances membrane permeability in Pm without compromising membrane integrity, dissipates membrane potential, increases intracellular doxycycline accumulation, and downregulates the transcription of the tetracycline efflux gene tet(B). Morphological analyses further revealed pronounced membrane deformation and possible leakage of intracellular contents. These findings highlight metformin as a potent, low-toxicity tetracycline adjuvant with cross-species efficacy, offering a promising therapeutic approach for managing tetracycline-resistant Pm infections. Full article

Vaccination plays a pivotal role in the control and prevention of animal infectious diseases. However, no efficient and safe universal vaccines are currently registered for major pathogens such as influenza A virus, foot-and-mouth disease virus (FMDV), simian immunodeficiency virus (SIV), and small ruminant lentiviruses (SRLV). Here, we review the development of Sendai virus (SeV) vectors as a promising vaccine platform for animal diseases. Recombinant SeV vectors (rSeVv) possess several key features that make them highly suitable for developing vaccination strategies: (1) SeV has exclusively cytoplasmic replication cycle, therefore incapable of transforming host cells by integrating into the cellular genome, (2) rSeVv can accommodate large foreign gene/s inserts (~5 kb) with strong but adjustable transgene expression, (3) can be propagated to high titers in both embryonated chicken eggs and mammalian cell lines, (4) exhibits potent infectivity across a broad range of mammalian cells from different animals species, (5) undergo transient replication in the upper and lower respiratory tracts of non-natural hosts, (6) has not been associated with disease in pigs, non-humans primates, and small ruminants, ensuring a favorable safety profile, and (7) induce a robust innate and cellular immune responses. Preclinical and clinical studies using rSeVv-based vaccines against influenza A virus, FMDV, SIV, and SRLV have yielded promising results. Therefore, this review highlights the potential of rSeVv-based vaccine platforms as a valuable strategy for combating animal viruses. Full article

As the incidence of tick-borne disease expands globally, comprehensive understanding of pathogen reservoir hosts is crucial to protect humans and wildlife. While many components are understood, there are gaps in our knowledge regarding the role of alternative, non-mammalian hosts such as birds. Within the United States, birds have been identified as reservoirs for Borrelia and Rickettsia; however, local studies rarely examine the potential of birds as reservoirs and transporters of Ehrlichia-infected ticks, unlike studies in Europe and South America. To address this research gap, we extracted and sequenced important microorganisms within 90 larval and nymphal ticks which were removed from passerine and near-passerine birds in the Great Plains region of the United States between May and October 2023. We found that 11% of birds hosted ticks infected with one or more Rickettsia or Ehrlichia species. Additionally, we collected a larval Haemaphysalis leporispalustris infected with Ehrlichia chaffeensis from a Northern Cardinal, the first North American songbird implicated in the Ehrlichia transmission cycle. Our research intertwines multiple bird and tick species in the North American pathogen system, highlighting the need for continued research focusing on birds as tick hosts and pathogen reservoirs in understudied parts of the United States. Full article

One of the most urgent threats to public health worldwide is the ongoing rise of multidrug-resistant (MDR) bacterial strains. Among the most critical pathogens are MDR-Klebsiella pneumoniae strains. The lack of new antibiotics has led to an increased need for non-antibiotic antimicrobial therapies. Photodynamic therapy (PDT) has become increasingly significant in treating MDR bacteria. PDT uses photosensitizer compounds (PS) that generate reactive oxygen species (ROS) when activated by light. These ROS produce localized oxidative stress, damaging the bacterial envelope. A downside of PDT is the limited bioavailability of PSs in vivo, which can be enhanced by conjugating them with carriers like nanoparticles (NPs). Zinc nanoparticles possess antibacterial properties, decreasing the adherence and viability of microorganisms on surfaces. The additive or synergistic effect of the combined NP-PS could improve phototherapeutic action. Therefore, this study evaluated the effectiveness of the copper(I)-based PS CuC1 compound in combination with Zinc Oxide NP, ZnONP, to inhibit the growth of both MDR and sensitive K. pneumoniae strains. The reduction in bacterial viability after exposure to a PS/NP mixture activated by 61.2 J/cm² of blue light photodynamic treatment was assessed. The optimal PS/NP ratio was determined at 2 µg/mL of CuC1 combined with 64 µg/mL of ZnONP as the minimum effective concentration (MEC). The bacterial gene response aligned with a mechanism of photooxidative stress induced by the treatment, which damages the bacterial cell envelope. Additionally, we found that the PS/NP mixture is not harmful to mammalian cells, such as Hep-G2 and HEK-293. In conclusion, the CuC1/ZnONP combination could effectively aid in enhancing the antimicrobial treatment of infections caused by MDR bacteria. Full article

SARS-CoV-2 is classified as a containment level 3 (CL3) pathogen, limiting research access and antiviral testing. To address this, we developed a non-infectious viral surrogate system using reverse genetics to generate sub-genomic replicons. These replicons contained the nsp1 mutations K164A and H165A and had the spike, membrane, ORF6, and ORF7a coding sequences replaced with various reporter and selectable marker genes. Replicons based on the ancestral Wuhan Hu-1 strain and the Delta variant of concern were replication-competent in multiple cell lines, as assessed by Renilla luciferase activity, fluorescence, immunofluorescence staining, and single-molecule fluorescent in situ hybridization. Antiviral assays using transient replicon expression showed that remdesivir effectively inhibited both replicon and viral replication. Ritonavir and cobicistat inhibited Delta variant replicons similarly to wild-type virus but did not inhibit Wuhan Hu-1 replicon replication. To further investigate the impact of nsp1 mutations, we generated a recombinant SARS-CoV-2 virus carrying the K164A and H165A mutations. The virus exhibited attenuated replication across a range of mammalian cell lines, was restricted by the type I interferon response, and showed reduced cytopathic effects. These findings highlight the utility of sub-genomic replicons as reliable CL2-compatible surrogates for studying SARS-CoV-2 replication and drug activity mechanisms. Full article

Cellular metabolism must adapt rapidly to environmental alterations and adjust nutrient uptake. Low glucose availability activates the AMP-dependent kinase (AMPK) pathway. We demonstrate that activation of AMPK or the downstream Unc-51-like autophagy-activating kinase (ULK1) inhibits receptor-mediated endocytosis. Beyond limiting dextran uptake, this activation prevents endocytic uptake of human pathogenic enveloped and non-enveloped, positive- and negative-stranded RNA viruses, such as yellow fever, dengue, tick-borne encephalitis, chikungunya, polio, rubella, rabies lyssavirus, and SARS-CoV-2, not only in mammalian and insect cells but also in precision-cut lung slices and neuronal organoids. ULK1 activation inhibited enveloped viruses but not EV71. However, receptor presentation at the cytoplasmic membrane remained unaffected, indicating that receptor binding was unchanged, while later stages of endocytosis were targeted via two distinct pathways. Drug-induced activation of the AMPK pathway reduced early endocytic factor TXNIP by suppressing translation. In contrast, the amounts of Rab5 and the late endosomal marker Rab7 decreased due to translation inactivation and ULK1-dependent proteasome activation within minutes. Furthermore, activation of AMPK hindered the late replication steps of SARS-CoV-2 by reducing viral RNAs and proteins and the endo-lysosomal markers LAMP1 and GRP78, suggesting a reduction in early and late endosomes and lysosomes. Inhibition of the PI3K and mTORC2 pathways, which sense amino acid and growth factor availability, promotes AMPK activity and blocks viral entry. Our results indicate that AMPK and ULK1 emerge as restriction factors of cellular endocytosis, impeding the receptor-mediated endocytic entry of enveloped and non-enveloped RNA viruses. Full article

The incidence of fungal infections continues to increase and one of the factors responsible for these high rates is the emergence of multi-resistant species, hospitalizations, inappropriate or prolonged use of medications, and pandemics, such as the ongoing HIV/AIDS pandemic. The recent pandemic caused by the severe acute respiratory syndrome virus (SARS-CoV-2) has led to a significant increase in fungal infections, especially systemic mycoses caused by opportunistic fungi. There is a growing and urgent need to better understand how these microorganisms cause infection and develop resistance as well as to develop new therapeutic strategies to combat the diverse diseases caused by fungi. Non-mammalian hosts are increasingly used as alternative models to study microbial infections. Due to their low cost, simplicity of care, conserved innate immunity and reduced ethical issues, the greater wax moth Galleria mellonella is an excellent model host for studying fungal infections and it is currently widely used to study fungal pathogenesis and develop innovative strategies to mitigate the mycoses studied. G. mellonella can grow at 37 °C, which is similar to the mammalian temperature, and the anatomy of the larvae allows researchers to easily deliver pathogens, biological products, compounds and drugs. The aim of this review is to describe how G. mellonella is being used as a model system to study fungal infections as well as the importance of this model in evaluating the antifungal profile of potential drug candidates or new therapies against fungi. Full article

The spread of multidrug-resistant microbes has made it necessary and urgent to develop new strategies to deal with the infections they cause. Some of these are based on nanotechnology, which has revolutionized many fields in medicine. Evaluating the safety and efficacy of these new antimicrobial strategies requires testing in animal models before being tested in clinical trials. In this context, Galleria mellonella could represent a valid alternative to traditional mammalian and non-mammalian animal models, due to its low cost, ease of handling, and valuable biological properties to investigate host–pathogen interactions. The purpose of this review is to provide an updated overview of the literature concerning the use of G. mellonella larvae as an animal model to evaluate safety and efficacy of nanoparticles and nanomaterials, particularly, of those that are used or are under investigation to combat microbial pathogens. Full article

The mammalian Apolipoprotein-L families (APOLs) contain several isoforms of membrane-interacting proteins, some of which are involved in the control of membrane dynamics (traffic, fission and fusion). Specifically, human APOL1 and APOL3 appear to control membrane remodeling linked to pathogen infection. Through its association with Non-Muscular Myosin-2A (NM2A), APOL1 controls Golgi-derived trafficking of vesicles carrying the lipid scramblase Autophagy-9A (ATG9A). These vesicles deliver APOL3 together with phosphatidylinositol-4-kinase-B (PI4KB) and activated Stimulator of Interferon Genes (STING) to mitochondrion–endoplasmic reticulum (ER) contact sites (MERCSs) for the induction and completion of mitophagy and apoptosis. Through direct interactions with PI4KB and PI4KB activity controllers (Neuronal Calcium Sensor-1, or NCS1, Calneuron-1, or CALN1, and ADP-Ribosylation Factor-1, or ARF1), APOL3 controls PI(4)P synthesis. PI(4)P is required for different processes linked to infection-induced inflammation: (i) STING activation at the Golgi and subsequent lysosomal degradation for inflammation termination; (ii) mitochondrion fission at MERCSs for induction of mitophagy and apoptosis; and (iii) phagolysosome formation for antigen processing. In addition, APOL3 governs mitophagosome fusion with endolysosomes for mitophagy completion, and the APOL3-like murine APOL7C is involved in phagosome permeabilization linked to antigen cross-presentation in dendritic cells. Similarly, APOL3 can induce the fusion of intracellular bacterial membranes, and a role in membrane fusion can also be proposed for endothelial APOLd1 and adipocyte mAPOL6, which promote angiogenesis and adipogenesis, respectively, under inflammatory conditions. Thus, different APOL isoforms play distinct roles in membrane remodeling associated with inflammation. Full article