2026-03-31
Moru Xu, Yupeng Liu, Xin Lan, Zhenyu Wang, Kun Qian, Hongxia Shao, Jinlin Huang, Jianqiang Ye, Aijian Qin
by Moru Xu, Yupeng Liu, Xin Lan, Zhenyu Wang, Kun Qian, Hongxia Shao, Jinlin Huang, Jianqiang Ye, Aijian Qin Many RNA viruses exhibit error-prone replication. Continuous generation of erroneous copies accelerates evolution. Avian leukosis virus subgroup J (ALV-J), an avian oncogenic virus, is a classical model virus for studying retroviruses. ALV-J's high mutation rates drive continuous evolution of its envelope and pathogenicity, posing significant challenges to the poultry industry. Here we employed deep mutational scanning to systematically assess envelope-wide mutation effects on ALV-J replication, integrating high-throughput sequencing with mutant libraries to identify critical envelope residues impacting viral fitness. Following 10 passages, the library virus exhibited enhanced replication capacity. Moreover, the library virus derived from SPF chickens displays screening results similar to those of the DF-1 cell-passaged virus. Most mutations were progressively eliminated during viral passaging, especially the first 80 amino acids of ALV-J envelope. Critical amino acid mutations, preferential deletion/insertion mutations and glycosylation patterns recapitulate evolutionary patterns observed in natural ALV-J isolates. Incorporation of all identified mutations into ALV-J J1 significantly increased in vivo replication efficiency and viral shedding of the recombinant virus. Functional study demonstrated that two key mutations independently promote viral replication: A64T enhancing entry via receptor-binding optimization, H304R promoting maturation through envelope cleavage efficiency. These insights enable targeted antiviral design by predicting evolutionary paths.
DOI: 10.1371/journal.ppat.10141102026-03-30
Max A. Odem, Sabona B. Simbassa, Cecilia Fadhel Alvarez, Oliyad Jeilu, Shelby R. Simar, Rachel Bosserman, Soumita Dutta, Walter Galdamez, Hossaena Ayele, Blake M. Hanson, Diana M. Proctor, Anne Marie Krachler
by Max A. Odem, Sabona B. Simbassa, Cecilia Fadhel Alvarez, Oliyad Jeilu, Shelby R. Simar, Rachel Bosserman, Soumita Dutta, Walter Galdamez, Hossaena Ayele, Blake M. Hanson, Diana M. Proctor, Anne Marie Krachler Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of food-borne illnesses, and disease severity correlates with the production of Shiga toxins. While clinical symptoms such as bloody diarrhea and haemolytic uremic syndrome have been attributed to Stx, its contribution to bacterial fitness is not well understood. Here, we demonstrate that Stx2 enhances STEC colonization of the zebrafish gut by facilitating the partial displacement of gut resident microbes. Infection with Stx2-producing STEC strains or direct exposure of fish to purified Stx2 induces alterations in the zebrafish microbiome structure, impacting several bacterial phyla and genera, notably Pseudomonads. We show that Stx2 is sufficient to facilitate these changes by accelerating intestinal transit, leading to increased expulsion of select gut microbes, including resident Pseudomonas species. Additionally, prokinetic drug treatment causes similar changes in gut transit and expulsion of Pseudomonas . Collectively, these findings detail a novel mode of action of Stx2 on the host, and shed light on its contribution to bacterial fitness within the host intestine.
DOI: 10.1371/journal.ppat.10141042026-03-27
Shankar Thangamani, Abishek Balakumar, Abhishek Datta, Garrett Bryak, Michail S. Lionakis
by Shankar Thangamani, Abishek Balakumar, Abhishek Datta, Garrett Bryak, Michail S. Lionakis Candida auris is an emerging, multidrug-resistant fungal pathogen that causes healthcare-associated outbreaks and life-threatening systemic infections. Unlike other Candida species, C. auris exhibits a distinct capacity for persistent skin colonization. In this review, we summarize our current understanding of clinical risk factors and host-microbe interactions that underlie C. auris skin colonization and infection. We discuss fungal determinants, including the unique mannan outer layer, fungal adhesins, the protein kinase Hog1, and other pathways in C. auris that govern adaptation in the skin. Furthermore, we highlight host immune mechanisms, including cytokine mediators (IL-1Ra, IL-17) and innate immune cells (neutrophils, macrophages, innate lymphocytes), that shape the outcome of C. auris skin colonization and infection. We also discuss how excessive IFN-γ responses drive epithelial pathology at the cutaneous barrier and enhance fungal persistence. Finally, we outline emerging research directions to understand host and microbe factors governing long-term colonization, with implications for developing novel therapeutic and vaccine strategies against this skin-tropic, multidrug-resistant fungal pathogen.
DOI: 10.1371/journal.ppat.10140752026-03-25
Soheila Javadi, Stephan Pienkoß, Dominik Meggers, Andrea Wimbert, Vivian B. Brandenburg, Pascal Dietze, Sina Schäkermann, Lilo Greune, Petra Dersch, Franz Narberhaus
by Soheila Javadi, Stephan Pienkoß, Dominik Meggers, Andrea Wimbert, Vivian B. Brandenburg, Pascal Dietze, Sina Schäkermann, Lilo Greune, Petra Dersch, Franz Narberhaus The nucleoid-associated protein Fis is a key transcriptional regulator in Gram-negative bacteria that supports rapid adaptation to environmental changes. In Yersinia pseudotuberculosis , Fis plays a critical, yet poorly understood role in virulence. Here, we present a comparative transcriptomic analysis of Y. pseudotuberculosis wild type and fis deletion mutant at environmental (25°C) and host-relevant (37°C) temperatures. Our data show that Fis modulates the expression of more than 600 genes across 16 functional categories. Notably, Fis exerts reciprocal, temperature-dependent control over virulence genes, including those encoding the type III secretion system (T3SS) and Yersinia effector proteins (Yops), flagella biosynthesis, and cell adherence/invasion factors. Functional assays revealed that fis deletion disrupts this regulatory balance, producing a host-defense-like state at 25°C characterized by complete loss of motility, upregulation of the virulence master regulator LcrF, aberrant Yop secretion, impaired phagocytosis by host cells, and increased pathogenicity in the Galleria mellonella infection model. These findings establish Fis as a central regulator that coordinates motility and early host-cell engagement while preventing premature activation of antiphagocytic defenses, thereby optimizing the initial stages of infection.
DOI: 10.1371/journal.ppat.10141052026-03-25
Melanie Ridgway, Douglas O. Escrivani, Markéta Novotná, Amy Wood, Michele Tinti, Achim Schnaufer, David Horn
by Melanie Ridgway, Douglas O. Escrivani, Markéta Novotná, Amy Wood, Michele Tinti, Achim Schnaufer, David Horn The kinetoplast incorporates the large mitochondrial genome present in the eponymous Kinetoplastida. Trypanosoma brucei is an African trypanosome that can lose kinetoplast DNA (kDNA), however, when the nuclear-encoded gamma subunit of the mitochondrial F 1 F O -ATP synthase (γATPase) is mutated. These mutations, analogous to a broken camshaft at the core of the ATP synthase rotary motor, are associated with multidrug resistance, and correlated with tsetse-fly independent mechanical transmission, and geographical spread of these parasites beyond Africa. Here we engineer kDNA-independent T. brucei to explore origins and consequences of kDNA loss. We use oligo targeting to edit the native γATPase gene, and selection with the ATP synthase targeting drug oligomycin to enrich the desired mutants. Using this approach, we identify novel M 282 F, M 282 W, and M 282 Y mutants, and subsequently generate precision-edited strains expressing the previously described L 262 P or A 273 P mutants, or the novel M 282 F mutant. Heterozygous M 282 F mutants retain sensitivity to the kDNA-targeting drug acriflavine, while homozygous M 282 F mutants are acriflavine resistant. Proteomic analysis of the kDNA-positive homozygous M 282 F mutant reveals highly specific depletion of ATP synthase-associated proteins, but not the F 1 subunits. Proteomic analysis following acriflavine-induced kDNA loss then reveals depletion of kDNA-binding proteins and mitochondrial RNA-processing factors alongside increased expression of mitochondrial membrane-associated transporters. We conclude that T. brucei cells with a homozygous γATPase M 282 F mutation remodel ATP synthase subunit expression and readily tolerate kDNA loss, which is accompanied by substantial remodelling of the mitochondrial proteome.
DOI: 10.1371/journal.ppat.10138462026-03-25
Yan Wang, Yanping Hu, Zhiqiang Ku, Jason Yeung, Jing Zou, Michael Woodson, Nikolai S. Prokhorov, Ekaterina S. Knyazhanskaya, Haiqing Zhao, Michael B. Sherman, Zhiqiang An, Stephen F. Carroll, Pei-Yong Shi, Petr G. Leiman, Xuping Xie
by Yan Wang, Yanping Hu, Zhiqiang Ku, Jason Yeung, Jing Zou, Michael Woodson, Nikolai S. Prokhorov, Ekaterina S. Knyazhanskaya, Haiqing Zhao, Michael B. Sherman, Zhiqiang An, Stephen F. Carroll, Pei-Yong Shi, Petr G. Leiman, Xuping Xie Engineered immunoglobulin M (IgM) antibodies typically exhibit superior neutralization potency and avidity compared to their parental IgG counterparts, primarily due to multivalent binding to repeated epitopes on a targeting antigen. In this study, we characterize the neutralization breadth and mechanism of action of IgM-14, a previously reported intranasally deliverable antibody targeting SARS-CoV-2. IgM-14 demonstrates remarkably potent antiviral activity against all pre-Omicron variants but significantly reduced efficacy against Omicron BA.1, and complete loss of activity against the later subvariant JN.1. Resistance selection identified two key mutations in the receptor-binding domain (RBD), G476D and F486P, which disrupt IgM-14 binding and confer strong resistance. Cryo-electron microscopy analysis uncovered two distinct Fab-RBD interfaces: a primary interface overlapping the angiotensin-converting enzyme 2 (ACE2)-binding region, and a unique secondary interface formed only when the RBD adopts the ACE2-inaccessible “down” conformation, involving a neighboring spike protomer. Site-directed mutagenesis and structural modeling revealed a critical role of this secondary site in IgM-14-mediated neutralization. Unlike IgG-14, structural modeling suggested that IgM-14 can simultaneously engage both interfaces in diverse modes, indicating a noncanonical avidity mechanism. Collectively, these findings highlight the structural and functional uniqueness of IgM-14 and offer valuable insights into the rational design of next-generation spike-targeted antibody therapeutics with enhanced breadth and potency.
DOI: 10.1371/journal.ppat.10140712026-03-23
Lauren E. Haynes, Ashley P. Barry, Micah A. Luftig
by Lauren E. Haynes, Ashley P. Barry, Micah A. Luftig Epstein-Barr virus (EBV) is associated with multiple malignancies including Burkitt lymphoma (BL), Hodgkin’s lymphomas, nasopharyngeal carcinomas (NPC), and gastric cancers. Canonically, EBV positive tumors display latent gene expression programs that are difficult to target pharmacologically. To overcome this hurdle, lytic reactivation therapies have been developed based on HDAC inhibition with limited mechanistic studies. We therefore characterized the impact of pan-HDAC inhibitor, panobinostat, and class I HDAC inhibitor, nanatinostat, on the growth, survival, and lytic reactivation of four EBV-positive cell lines: P3HR1-ZHT BL, Jijoye BL, IBL-1 immunoblastic lymphoma, and de novo infection derived lymphoblastoid cell lines (LCL). All lines were sensitive, enabling us to define ranges of sensitivity within which to use single cell approaches to assess early EBV lytic gene expression, cell cycle state, and apoptosis. We observed that each EBV-positive model of malignancy responded uniquely to the same HDAC inhibitors and that lytic reactivation was successful in only a small percentage of the cell population. To elucidate the potential role of host factors in preventing successful lytic reactivation, we performed single-cell RNA sequencing on the P3HR1-ZHT BL line treated with the HDAC inhibitor panobinostat. We observed that abortive lytic cells, or cells that do not successfully progress through the lytic cycle, upregulated genes downstream of NF-κB activity. Additionally, genes involved in immune signaling including the CD137/CD137L signaling axis, were upregulated in abortive lytic cells. Functional validation through a Cas9-RNP approach revealed that the CD137 receptor is indeed involved in preventing successful lytic reactivation. These data have important implications for how we approach oncolytic therapies for EBV-associated malignancies.
DOI: 10.1371/journal.ppat.10136102026-03-23
Conor W. Templeton, Jasmine S. Gulik, Laimonis A. Laimins
by Conor W. Templeton, Jasmine S. Gulik, Laimonis A. Laimins APOBECs are cytidine deaminases whose levels are increased in cells with high-risk HPV genomes and are responsible for most mutations in HPV associated cancers. APOBEC3B is a nuclear member of this family and is shown to be a positive regulator of HPV replication as well as expression. The proviral effects of A3B found in HPV positive cells contrast with its role as a restriction factor for many other viruses. Studies demonstrated that A3B can bind and regulate the formation of R-loops, which are trimeric nucleic acid structures consisting of an RNA paired with its complementary DNA strand, displacing one of the DNA strands. The present study demonstrates that A3B binds stably to both cellular and viral chromatin at sequences containing high R-loop levels, including the HPV URR and early polyadenylation sites. Importantly, A3B was found to play a critical role in the replication of HPV genomes and in regulating viral expression. Reduction of R-loop levels through overexpression of the R-loop specific RNase, RNase H1, impaired A3B binding to viral genomes as well as at multiple cellular sites. When A3B was depleted, total R-loop levels decreased by ~50%, leading to impaired viral transcription and an increase in the expression of immune genes, such as OASL, IL6, and IRF1. Mapping R-loop formation in A3B depleted cells revealed that A3B regulated a subset of R-loops that form on the transcriptional start (TSS) and termination sites (TTS) of cellular genes, including at the HPV URR. Furthermore, A3B depletion resulted in over a 50% reduction of DNA breaks along with altered expression of DNA damage repair proteins. This study demonstrates that A3B is an inducer of R-loop formation and DNA damage in HPV positive cells, thereby regulating cellular and viral gene expression along with HPV replication.
DOI: 10.1371/journal.ppat.10140882026-03-23
Ziyun Zou, Yibo Hu, Zhongxia Guan, Jiajing Chen, Qingyao Zhang, Yinze Han, Junyu Zhu, Chunxia Wang, Bing Han, Tian Li, Zeyang Zhou
by Ziyun Zou, Yibo Hu, Zhongxia Guan, Jiajing Chen, Qingyao Zhang, Yinze Han, Junyu Zhu, Chunxia Wang, Bing Han, Tian Li, Zeyang Zhou Microsporidia are known intracellular pathogens that infect nearly all animals and deeply manipulate host mitochondrial homeostasis for survival. Here, we report a novel mechanism by which the human-pathogenic Encephalitozoon hellem modulates the mitophagy machinery of its host. We identified the secreted protein EhPTP4 as a key effector in disrupting selective degradation processes in the infected cells. EhPTP4 is found to localize within the nucleus of infected cells, where it induces increased expression of endoplasmic reticulum-associated degradation (ERAD) pathway components, including HSPA5, HERPUD1, and PDIA4. This induction enhances protein ubiquitination in host cells and leads to the degradation of BNIP3L, a critical regulator of mitophagy. Investigation into the molecular interaction network revealed that EhPTP4 interacts with host corepressor RCOR1 and histone H3. This interaction modulates histone acetylation, specifically at H3K14ac sites, thereby further influencing the expression of a key ERAD gene, HERPUD1. This study uncovers a sophisticated strategy by which microsporidia manipulates both ER stress response and the histone acetylation to suppress mitophagy. These findings provide new insights into the mechanisms of microsporidian pathogenesis.
DOI: 10.1371/journal.ppat.10140782026-03-23
Clara Blancfuney, Eva Guchen, Marie Garcia, Julien Faccini, Jean-François Sutra, Felipe Ramon-Portugal, Elise Courtot, Marlène Z. Lacroix, Roger Prichard, Anne Lespine, Mélanie Alberich
by Clara Blancfuney, Eva Guchen, Marie Garcia, Julien Faccini, Jean-François Sutra, Felipe Ramon-Portugal, Elise Courtot, Marlène Z. Lacroix, Roger Prichard, Anne Lespine, Mélanie Alberich Helminth infections in grazing ruminants are of major concern for animal welfare and cause substantial economic losses, prompting the widespread use of ivermectin (IVM). The emergence of IVM resistance, driven by complex and poorly understood mechanisms, increasingly compromises treatment efficacy. Drug efflux transporters, particularly P-glycoproteins (PGPs), are suspected to contribute to resistance. Yet, the study of their individual and functional role is hindered by their diversity in nematodes. This study aimed to dissect the role of specific PGPs in mediating IVM resistance. Thus, the Caenorhabditis elegans strain IVR10, selected for IVM resistance and reported to overexpress pgp s, was used as a model. We generated different IVR10 strains each lacking one of six key pgp s, and assessed changes in IVM tolerance. Remarkably, only the deletion of pgp-9 significantly increased IVM sensitivity. Furthermore, transgenic expression of Haemonchus contortus pgp-9.1 rescued the resistant phenotype, demonstrating a conserved function across species. To explore drug dynamics, we developed a fluorescent IVM analog, which revealed reduced drug accumulation in IVR10, a phenotype reversed by pgp-9 deletion. Altogether, these findings show that nematode PGP-9 modulates IVM tolerance in IVR10 by controlling drug efflux and highlight it as a potential therapeutic target.
DOI: 10.1371/journal.ppat.10133552026-03-23
Jelle M. Melchers, Jarek Juraszek, Ruben J.G. Hulswit, Daan van Overveld, Lam Le, Frank J.M. van Kuppeveld, Daniel L. Hurdiss, Berend-Jan Bosch, Johannes P.M. Langedijk, Mark J.G. Bakkers
by Jelle M. Melchers, Jarek Juraszek, Ruben J.G. Hulswit, Daan van Overveld, Lam Le, Frank J.M. van Kuppeveld, Daniel L. Hurdiss, Berend-Jan Bosch, Johannes P.M. Langedijk, Mark J.G. Bakkers The continued threat of zoonotic coronavirus spillovers underscores the need for cross-species applicable vaccine design strategies. The genus Embecovirus includes human coronaviruses OC43 and HKU1 as well as relevant veterinary pathogens. The coronavirus spike (S) fusion glycoprotein, key to viral entry and protective immunity, is inherently metastable, complicating vaccine development. Using the ReCaP AI tool, we stabilized the prefusion conformation of OC43 S through rationally combined amino acid substitutions, resulting in markedly enhanced expression and thermal stability. The substitutions were transferable to equine coronavirus (ECoV) S and HKU1. Cryo-EM structures of stabilized OC43 and ECoV S revealed that stabilization was achieved by arresting the release of the fusion peptide and keeping the S1 B receptor binding domain in the ‘down’ state by improving the complex polar interactions of neighboring S1 B domains and the bound free fatty acid at the interprotomer S1 B interface. This work provides the first ECoV S structure and a broadly applicable framework for engineering stabilized Embecovirus S antigens.
DOI: 10.1371/journal.ppat.1013998