PLoS Pathogens最新文献

African swine fever virus (ASFV) infection causes a severe hemorrhagic disease in pigs, characterized by excessive inflammatory responses and tissue damage, posing substantial threats to the pig industry worldwide. Given the lack of vaccines and effective antiviral treatments, as well as the largely unknown functions of most ASFV-encoded proteins, it's urgent to study the proteins that are crucial in triggering inflammatory responses and how they do so. This study demonstrated that ASFV exploited the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome to induce pyroptosis and inflammatory responses, effectively replacing the non-functional porcine AIM2 pseudogene. Screening over 150 proteins encoded by the ASFV genome, EP364R was identified as the viral factor responsible for driving NLRP3-mediated pyroptosis and high-level cytokine production. Ectopic expression of EP364R in mice elicited significant upregulation of serum pro-inflammatory cytokines and splenomegaly, while its expression in bone marrow-derived macrophages (BMDMs) from NLRP3-knockout mice abrogated pyroptosis and related effects. Mechanistic investigation revealed that the helicase DDX3X acted as a molecular bridge, enabling EP364R to interact with NLRP3 to promote the aggregation and activation of inflammasomes. Depletion of DDX3X abolished EP364R's ability to induce NLRP3-dependent pyroptosis and pro-inflammatory cytokine production. We found that the NACHT domain of porcine NLRP3 interacted with DDX3X, and EP364R established a connection with the NACHT and LRR domains of NLRP3 through DDX3X. However, EP364R bound to all the domains of DDX3X. Molecular docking analysis revealed that DDX3X interacted with EP364R through a spatially defined interface, thereby exerting its function. Furthermore, a natural compound library was employed to screen functional compounds targeting EP364R, and HAMNO was identified as an inhibitor that bound to E256, K259, and D260 of EP364R, consequently suppressing ASFV replication. Our findings explain how ASFV triggers pyroptosis and excessive cytokine release, and identify a potent small-molecule inhibitor of ASFV, aiding the development of vaccines and therapies to prevent and control African swine fever (ASF) caused by ASFV infection.

Leishmania spp. are intracellular parasites that cause leishmaniasis, a devastating disease with no effective treatment. These parasites are heme auxotrophs and must scavenge this essential cofactor from the host. Transcriptomic analysis of Leishmania major promastigotes cultured in the presence or absence of heme revealed numerous differentially expressed genes. Among those of unknown function, LHR2 (Leishmania Heme Response-2) was the most upregulated gene in response to heme limitation. LHR2 encodes a mitochondrial hemoprotein that likely protects this organelle from elevated levels of reactive oxygen species. It is essential during the promastigote stage, and loss of a single LHR2 allele severely compromises intracellular replication and prevents the development of cutaneous leishmaniasis in mice. This essential function depends on LHR2's ability to bind heme. Complementation studies in Saccharomyces cerevisiae revealed that LHR2 is an analogue of the yeast Dap1p, although it binds heme in a distinct manner. Importantly, LHR2 displays key structural differences from the most closely related human proteins. These findings highlight LHR2 as a critical factor in parasite survival and pathogenesis, and suggest it as a promising new target for antileishmanial drug development.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) poses a significant zoonotic risk. The absence of the structure of SADS-CoV main protease (Mpro) severely impedes the development of effective antiviral therapeutics. Here, we present the high-resolution structures of SADS-CoV Mpro and its complexes with inhibitors 27h and SY110, respectively. These two compounds inhibit SADS-CoV Mpro through a novel inhibition mechanism. Residues 40-53 of SADS-CoV Mpro adopt a single-helix conformation, in contrast to a coiled coil formed by two consecutive alpha-helices observed in SARS-CoV-2 Mpro. These structural differences contribute to the varying inhibitor potency between Alphacoronavirus (α-CoV) and Betacoronavirus (β-CoV) Mpros. We subsequently demonstrate that the absence of residue '51' in α-CoV Mpros plays a key role in these conformational changes. Furthermore, 27h was proved to efficiently suppress SADS-CoV replication in both cell-based assays and porcine intestinal organoids-marking the first such assessment. Overall, these findings reveal that intrinsic Mpro dynamics influence inhibitor potency and provide insights for designing broad-spectrum Mpro inhibitors.

Background and aims: Hepatitis B virus (HBV) replication generates a double-stranded linear DNA (dslDNA) byproduct. This dslDNA can undergo intermolecular and intramolecular nonhomologous end-joining (NHEJ) recombination, resulting in viral integration and dslDNA-derived covalently closed circular DNAs (dsl-cccDNAs), respectively. The insertions and deletions (INDELs) at the end-joining site have been used to differentiate dsl-cccDNA from the authentic cccDNA. The prevalence and characteristics of dsl-cccDNA in chronic hepatitis B (CHB) patients remain unclear.

Approach and results: HBV-targeted next-generation sequencing (NGS) was used to identify 32 dsl-cccDNA-positive candidates, 22 HBeAg(+) and 10 HBeAg(-), from 56 liver biopsies of antiviral treatment-naïve CHB patients for dsl-cccDNA confirmation and characterization by PSAD-cccDNA PCR NGS. INDELs within the DR2-1 region (nt 1600-1840) of the cccDNA were analyzed. Various clonally expanded, heterogenous ~22-nt deletions in the X gene region around nt 1760 were discovered in all 32 samples. The dsl-cccDNA species were then defined and characterized by the INDELs clustered at the DR1 surrounding region (nt 1800-1840). The proportion of dsl-cccDNA in total cccDNA was higher among HBeAg(+) compared to HBeAg(-) samples. The diversity of dsl-cccDNA species positively correlated with cccDNA levels and serum viral load, and was higher in HBeAg(+) CHB.

Conclusions: dsl-cccDNA is more abundant and diverse among the HBeAg(+) CHB subjects. The existence of replication-defective dsl-cccDNA may facilitate immune evasion and HBV integration, and complicate HBV pathogenesis.

Intestinal amoebiasis is caused by Entamoeba histolytica, one of the deadliest human-infective parasites. Central to its pathogenicity is its binding to mucosal carbohydrates, which precedes tissue damage by trogocytosis. Carbohydrate binding is mediated by a single adhesin, the galactose/N-acetylgalactosamine (Gal/GalNAc) lectin, which is the leading vaccine candidate for amoebiasis. We present the structure of the native heterodimeric lectin, revealing an ordered core containing the light chain and the N-terminal region of the heavy chain. Structures obtained in the presence of ligand show that the Gal/GalNAc binding site is in the light chain, which adopts a β-trefoil fold found in other lectins. An elongated arm emerges from the heavy chain, which adopts multiple positions and may be modulated by sugar binding. This study reveals the molecular basis for sugar binding by the Entamoeba histolytica Gal/GalNAc lectin, a prerequisite for parasite invasion and development of intestinal disease.

Commercial beekeepers in the US reported severe colony losses early in 2025, as colonies were being staged for their critical role in the almond pollination season in California. Average reported losses since the preceding spring exceeded 60%, with substantial variation among operations. Many colonies were still actively collapsing in January 2025, at which time pooled and individual samples were collected and then screened for levels of 13 known honey bee pathogens and parasites. Acute bee paralysis virus and other known viral pathogens were found at high levels in pooled bee samples from all collapsing apiaries. Nevertheless, viral loads did not differ between healthy colonies and colonies in active collapse. However, individual bees exhibiting shaking behaviors and morbidity showed distinctly higher loads of two strains of deformed wing virus. Differences between these two analyses suggest that direct collections of morbid bees provide a complementary diagnostic for causal viruses, a suggestion supported by inoculation experiments that successfully replicated observed pathologies. Since these viruses are known to be vectored by the parasitic mite Varroa destructor, mites from collapsed colonies were in turn screened for resistance to amitraz, a critical miticide used widely by beekeepers, including all beekeepers surveyed in this study. A genetic trait linked with miticide resistance was found in all collected mites, underscoring the urgent need for new control strategies for this parasite. While viruses are a likely end-stage cause of colony death, other stressors such as nutritional stress and agrochemicals may have also played significant roles.

Pore-forming toxins (PFTs) are key bacterial virulence factors that disrupt host plasma membrane (PM) integrity, triggering cellular stress and initiating repair mechanisms. The cytolysin Listeriolysin O (LLO), secreted by Listeria monocytogenes, has well established roles in infection, yet the host signaling responses to LLO-induced damage remain poorly understood. Here, we identify a previously unrecognized protective pathway in which LLO triggers rapid activation of the tyrosine kinase Src, leading to phosphorylation of the non-muscle myosin II heavy chain 2A (NMHC2A) at tyrosine 158. While Src activation and NMHC2A tyrosine phosphorylation have been observed during Listeria infection, we demonstrate here that both responses are directly driven by LLO. This phosphorylation event does not alter NMHC2A motor activity in vitro but is required for cytoskeletal reorganization and efficient responses to PM damage. Using Caenorhabditis elegans, we further show that phosphorylation of the NMHC2A homolog NMY-2 at the conserved tyrosine 163 is required for survival under PFT-induced stress and heat shock, revealing an evolutionarily conserved defense mechanism. Together, our findings establish Src-mediated NMHC2A phosphorylation as a critical link between PFT-induced PM damage sensing and actomyosin remodeling, advancing our understanding of host defense against bacterial toxins.

[This corrects the article DOI: 10.1371/journal.ppat.1012618.].

The currently licensed anti-tuberculosis (TB) vaccine, Mycobacterium bovis BCG, provides limited protection against pulmonary TB in adolescents and adults, the main cause of TB transmission and mortality. To obtain an improved BCG-based vaccine candidate with increased immune signalling but still low virulence, we have previously generated a recombinant BCG strain named BCG::ESX-1Mmar, which is heterologously expressing ESX-1 functions of Mycobacterium marinum and thereby modulates the host innate immune responses via phagosomal rupture-associated induction of type I interferon production and enhanced inflammasome activity, leading to superior protection against TB disease in murine infection models. As protection may also vary with the route of vaccination, here, we have explored aerosol vaccination relative to subcutaneous vaccination, using BCG Pasteur and BCG::ESX-1Mmar. We found that mice vaccinated via the aerosol route with BCG Pasteur or BCG::ESX-1Mmar both yielded higher frequencies of CD4+ and CD8+ Th1 activated effectors and T effector memory cells in the lungs compared to subcutaneously immunised mice, whereas comparable polyfunctional Th1 (IL-2, TNF-α and IFN-γ) cytokine-producing subsets were observed in the spleens of all vaccinated mice. Significantly higher IL-17A responses without severe lung pathology were seen in the lungs of aerosol-vaccinated mice associated to local and transient inflammatory cytokine responses and immune cell infiltrations. In contrast to the subcutaneous route, aerosol vaccination elicited high amounts of humoral IgG and IgA responses in the bronchoalveolar lavage fluid and induced a substantial number of lung CD4+ and CD8+ T cells expressing CD69+ CD103+ tissue-residency markers. These effects led to significant improved protection against M. tuberculosis and reduced lung pathology in aerosol-vaccinated mice compared to subcutaneously vaccinated mice. Moreover, BCG::ESX-1Mmar vaccine induced enhanced T-cell immunity and superior protection compared to parental BCG Pasteur for both vaccination routes and thereby represents an interesting candidate for developing improved vaccination strategies against TB.

Ebola virus (EBOV) is likely a zoonotic and re-emerging virus that causes severe outbreaks of Ebola virus disease. The virus spreads to various tissues during the late stage of infection and has been detected in immune-privileged sites of survivors. However, the mechanism of how EBOV disseminates throughout the body is not completely elucidated. In this study, by using a biologically contained EBOVΔVP30 system, we demonstrate that a megakaryocytic-like MEG-01 cell line that stably expresses VP30 (MEG-01 VP30 cells) is susceptible to EBOVΔVP30 infection and that MEG-01 VP30 cells exposed to EBOVΔVP30 produce platelet-like particles (PLPs) that contain EBOV proteins and viral genetic material. We further found that the viral envelope glycoprotein is expressed on the surface of the produced PLPs and contributes to PLP internalization into recipient cells. In addition, viral mRNA and genome RNA are actively synthesized in these PLPs, which may lead to progeny EBOV production from recipient cells that internalize the PLPs. Taken together, our data provide new insights into the potential role of platelets in the widespread dissemination of EBOV and the pathogenesis of Ebola virus disease.