Flaviviruses are enveloped, positive-strand RNA viruses that cause millions of infections in the human population annually. Although Zika virus (ZIKV) had been detected in humans as early as the 1950s, its reemergence in South America in 2015 resulted in a global health crisis. While flaviviruses encode 10 proteins that can be post-translationally modified by host enzymes, little is known regarding post-translational modifications (PTMs) of the flavivirus proteome. We used mass spectrometry to comprehensively identify host-driven PTMs on the ZIKV proteome. This approach allowed us to identify 43 PTMs across 8 ZIKV proteins, including several that are highly conserved within the Flavivirus genus. Notably, we found two phosphosites on the ZIKV envelope protein that are functionally important for viral propagation. Both appear to regulate viral particle release, while one also impacts ZIKV cytopathogenicity. Additionally, we discovered host kinases that interact with ZIKV proteins and determined that Bosutinib-an FDA-approved tyrosine kinase inhibitor that targets some of these host kinases-impairs ZIKV growth, in part by blocking phosphorylation of a tyrosine residue on the envelope protein. Thus, we have defined a high-resolution map of host-driven PTMs on ZIKV proteins as well as cellular interacting kinases, uncovered novel mechanisms of host driven-regulation of ZIKV particle release and cytopathogenicity, and identified an FDA-approved inhibitor of ZIKV growth.
Citrobacter rodentium (CR) is a murine-specific enteric pathogen widely used to model infections of the attaching and effacing pathogens enteropathogenic and enterohaemorrhagic Escherichia coli. Infection induces colonic epithelial damage and inflammation that are resolved in resistant mice but result in severe disease and mortality in mice lacking interleukin-22 (IL-22). While IL-22-deficient (Il22-/-) mice succumb to CR infection due to dehydration, in part mediated by disruption of the barrier integrity by the type III secretion system (T3SS) effector EspF, the contribution of immunopathology to infection outcomes is not known. Here, using CR we tested whether dysregulated neutrophil responses drive disease progression in Il22-/- mice. We show that IL-22 deficiency is associated with progressive colonic pathology and increased neutrophil accumulation and activity in the colonic mucosa. Infection of mice with CR lacking the T3SS effector EspO, which is implicated in neutrophil recruitment, resulted in markedly reduced neutrophil accumulation and activity in Il22-/- mice without altering bacterial burden. Notably, CRΔespO infection led to reduced colonic inflammation and complete survival of Il22-/- mice. Consistent with these findings, CXCR2 antagonist treated Il22-/- mice displayed attenuated disease severity and delayed mortality. Together, we identified EspO as the second effector, alongside EspF, whose deletion results in survival of CR-infected Il22-/- mice. Moreover, these findings suggest that excessive neutrophil accumulation and activity are key drivers of lethality during CR infection in Il22-/- mice and that EspO amplifies neutrophil-dependent pathology within a vulnerable epithelial environment.
BACKGROUND:The rapid global spread of hypervirulence in Enterobacteriaceae, particularly in carbapenem-resistant Klebsiella pneumoniae, poses a significant public health threat. However, the key genetic vehicles and mechanisms driving horizontal transfer of hypervirulence-associated genes (iucA, iroB, rmpA, rmpA2, and peg-344) remain poorly defined, limiting effective surveillance. METHODS:We performed a large-scale genomic survey of 2,869 virulence-associated plasmid sequences and 2,337 complete Enterobacteriaceae chromosomes. Using comparative genomics and evolutionary analyses, we systematically identified and characterized Hypervirulence-associated Pseudo-Compound Transposons (Hva-PCTs), defined as structured mobile elements in which hypervirulence-associated genes are flanked by insertion sequences. RESULTS:Our results demonstrate that hypervirulence-associated genes are transmitted primarily as discrete IS-bounded units, which we term Hva-PCTs. We identified 29 distinct plasmid-borne Hva-PCTs (pHva-PCTs) and 30 chromosomal Hva-PCTs (cHva-PCTs). These modules show clear species-specific patterns: iucA/iroB-associated Hva-PCTs mainly originate in Escherichia coli and spread through IncFIB-containing multi-replicon plasmids (commonly combined with IncFIC(FII) and/or IncFII, while rmpA/rmpA2/peg-344-containing modules originate in K. pneumoniae and are disseminated via IncHI1B/repB plasmids. Three Hva-PCTs were detected on both plasmids and chromosomes (xHva-PCTs). In one clinical K. pneumoniae isolate (LS356), the identical composite module was present on both replicons. Simpler sub-modules, such as ISKqu3-rmpA2-iucA_1-IS102 and IS102-rmpA-peg-344-iroB_1-IS1A, frequently co-occur on the same plasmid; when positioned in tandem, they reconstitute the full composite structure. This assembly pattern is further supported by a partial duplication event in plasmid pP901. CD-HIT clustering (80% nucleotide identity and 90% coverage) showed that 13 of 22 major clusters contained both plasmid and chromosomal copies, with intra-cluster identities >80% across multiple sequence types and host species. CONCLUSION:Hypervirulence-associated genes in Enterobacteriaceae are disseminated mainly as IS-flanked Hva-PCTs rather than solely through intact virulence plasmids. These modules exhibit strong but not absolute host specificity. The presence of identical Hva-PCTs on plasmids and chromosomes suggests inter-replicon mobility, while their stepwise assembly from simpler sub-modules highlights modular accretion as a key evolutionary process. Tracking Hva-PCTs as distinct mobile units may complement existing plasmid- and gene-centric surveillance approaches for hypervirulent and convergent strains. Experimental validation of their transposition activity and phenotypic effects is still required.
Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a bat-originated alphacoronavirus that causes devastating enteric disease in neonatal piglets and possesses significant potential for cross-species transmission. While the early stages of the coronavirus life cycle have been extensively characterized, the host factors indispensable for virion assembly and subsequent export remain largely enigmatic. Here, by performing a genome-wide CRISPR-Cas9 knockout screen using a recombinant icSADS-CoV-GFP reporter virus, we identified the small GTPase Rab10 as a critical host dependency factor for SADS-CoV infection. Viral life cycle analysis revealed that Rab10 is not required for viral attachment, entry, or initial genome replication, but is essential for the virion transport and non-lytic egress. Rab10 deficiency markedly reduced the extracellular release of viral RNA, viral proteins, and infectious progeny, as well as the secretion of SADS-CoV virus-like particles. Confocal imaging showed that Rab10 and viral protein-positive intracellular structures were associated with LMAN1, TGN46, and LAMP1 positive compartments. These findings support a model in which Rab10 coordinates a virus-containing vesicles trafficking pathway associated with ERGIC-TGN-lysosome compartments. Mechanistically, Rab10 facilitates the loading of the viral envelope (E) protein into transport vesicles derived from the ERGIC. Rab10 associates with the SADS-CoV E protein, and mapping analyses implicated the C-terminal PDZ-binding motif, particularly residue V75, in efficient Rab10 association and viral release. Collectively, our findings identify Rab10 as a host regulator of SADS-CoV non-lytic egress and highlight the E-Rab10 interaction and the vesicular trafficking mechinery as a potential target for developing antiviral strategies.
Staphylococcus aureus α-toxin (AT) is a potent pore-forming toxin that causes cell damage during infection. The structure and activity of AT have been studied extensively, but the environmental factors that modulate its toxicity remain unclear. Here, we demonstrate that the extracellular pH modulates AT cytotoxicity through two independent mechanisms in human cells. First, we show that acidic conditions enhance AT pore maturation, causing a toxin-intrinsic and ADAM10 independent increase in cytotoxicity after prolonged exposure to AT. Second, we demonstrate that both acidic and alkaline conditions impair the cellular capacity to internalize AT pores, resulting in toxin retention at the cell surface and increased cytotoxicity after pulse intoxication. The cell-intrinsic, pH-dependent sensitization to AT is observed in various human cell types but not for other pore forming toxins. Together, our findings indicate that the extracellular pH modulates AT activity and host cell susceptibility.
TRAF6 is traditionally recognized as an antiviral ubiquitin E3 ligase that positively regulates the production of type I interferon and inflammatory cytokines. However, our study reveals that TRAF6 also plays a crucial role in the lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV). Mechanistically, during KSHV lytic replication, TRAF6 mediates the K63-linked polyubiquitination and activation of Akt, which is required for the efficient viral replication. Disruption of TRAF6 or Akt expression through CRISPR-mediated knockout, or inhibition of TRAF6 or Akt with small molecule inhibitors, reduces KSHV replication efficiency. Conversely, expression of constitutively active Akt can rescue the impaired replication caused by TRAF6 deficiency. Notably, the TRAF6-Akt axis is also required for the lytic replication of Epstein-Barr virus but not for Human cytomegalovirus. These findings highlight the role of the TRAF6-Akt axis in the life cycle of oncogenic herpesviruses and suggest potential therapeutic targets for related diseases.
Two-component systems (TCSs) in Pseudomonas aeruginosa are essential for sensing and responding to diverse environmental cues, including those associated with ion homeostasis and virulence within the host. However, how specific TCSs discriminate between chemically similar metal signals to fine-tune adaptation remains unclear. Here, we identify the TCS ColRS, conserved across the Pseudomonas genus, as a critical sensor that distinguishes zinc (Zn2+) and copper (Cu2+) to drive divergent survival strategies. First, we establish that the response regulator ColR directly binds the promoters of lipid A modification genes (arnB, eptA), the metal responsive regulator czcR, and the alginate and motility regulator Z (amrZ), genetically linking metal sensing to resistance and virulence circuitry. Phenotypically, ColRS translates these inputs into context-dependent states: enhancing polymyxin tolerance and suppressing motility under copper stress, while sustaining type III secretion system (T3SS) virulence and motility under zinc stress. Mechanistically, this discrimination relies on the sensor kinase ColS, which utilizes a non-canonical E96-H105 interface as a specificity filter to recognize Cu2+ (Kd ≈ 1.11 μM) and distinguish it from Zn2+ (Kd ≈ 19.8 μM). Global proteomics confirms this bifurcation, revealing that Zn2+ triggers focused metabolic tuning, whereas Cu2+ induces broad envelope and motility rewiring. Collectively, our findings reveal ColRS as a pivotal molecular hub that decodes the duality of host metal immunity to optimize pathoadaptation.
Cancer hallmarks are characterized by wide-scale changes in gene expression programs. Pioneering studies showed how viral oncogenes target regulatory pathways, but little is known about tumorigenic mechanisms of non-viral pathogens. Theileria annulata is an intracellular parasite (related to apicomplexa parasites causing malaria) which remarkably transforms bovine leukocytes, hijacking host signaling pathways to induce cancer phenotypes, akin to human leukemias. While some host genes contribute to the proliferative or invasive hallmark phenotypes, there is still limited comprehensive understanding of the impact of Theileria infection on host transcription and transformation. We performed a multi-omics meta-analysis to investigate the effect of Theileria infection on cancer hallmarks in bovine B lymphocytes. Combining transcriptomic, proteomic and epigenomic analysis across multiple datasets, we show that Theileria infection suppresses host immune pathways. Specifically, genes encoding innate and adaptive immune mediators are repressed in T. annulata-infected B cells (and in T. parva infected T cells), including downregulation of genes for Toll-like receptors (TLR), inflammasome components of the guanylate binding protein (GBP) family and major histocompatibility complex class (MHC) II genes. Treatment with distinct theilericidal drugs could partially rescue immune gene expression. Mechanistically, we describe alterations in the host epigenome, including loss of activating histone modifications (e.g., H3K18ac, H3K4me3, H3K27ac) on the promoters of repressed immune genes, and enrichment of silencing marks (H3K27me3) on promoters of the BOLA genes and the gene encoding CIITA, the master transcriptional regulator of MHC class II gene expression. Our results suggest that intracellular T. annulata and T. parva parasites could drive an immune evasion cancer hallmark in host lymphocytes by epigenetic silencing of genes for innate and adaptive immunity.
[This corrects the article DOI: 10.1371/journal.ppat.1012176.].
Malaria in pregnancy remains a major global health concern, contributing significantly to maternal and offspring morbidity and mortality. While gut microbiota dysregulation has been implicated in pregnancy complications and malaria pathogenesis, its functional role and the underlying mechanisms within the gut-placenta axis during placental malaria remain poorly understood. In pregnant mice, Plasmodium berghei ANKA infection disrupted the gut-placenta axis, leading to intestinal inflammation, placental injury and reduced fetal weight. Microbiome analysis revealed gut dysbiosis characterized by reduced abundance of Ligilactobacillus and increased abundance of Desulfovibrio. The metabolomic profiling identified disruption in amino acid and fatty acid metabolism, including changes in metabolites such as indole-3-propionic acid and taurine. These microbial and metabolic alterations may contribute to impaired intestinal barrier integrity and dysregulated inflammatory responses. Importantly, fecal microbiota transplantation (FMT) restored gut microbial balance, alleviated colonic and placental inflammation, and improved offspring growth. These findings provide novel mechanistic insights into the gut-placenta axis in malaria during pregnancy. Future studies should validate these findings in clinical settings and explore alternative microbiota-targeted interventions.
The malaria parasite Plasmodium falciparum is an obligate intracellular organism that spends an important part of its lifecycle inside human erythrocytes. The endocytosis of host-cell cytosol and its delivery to a lysosome-like organelle called the food vacuole are critical for the parasite’s survival and proliferation. Recent work has started to identify some of the molecular players involved in this pathway, but much remains to be discovered. Evidence suggests that phosphatidylinositol-3-phosphate (PI3P) plays a central role in this process. In unicellular eukaryotes, such as yeast, PI3P is generated by a single PI3-kinase, whose activity is regulated by a pseudokinase called Vps15. P. falciparum also possesses a PI3K that generates PI3P and bioinformatics analysis has revealed the presence of an uncharacterized putative orthologue of Vps15. We here present our characterization of PfVps15. We first show that it is constitutively expressed throughout the asexual erythrocytic cycle and that it interacts with PfPI3K, but unlike in yeast and mammalian cells, it is potentially not part of a heterotetrameric complex. The removal of PfVps15 from its site of action by knock sideways led to rapid parasite death. Phenotypic analyses revealed a decrease in PI3P levels, the abrogation of the delivery of host-cell cytosol containing vesicles to the food vacuole, and defects in apicoplast biogenesis and mitochondrial fission. Collectively, our data has identified a protein critical for the synthesis of PI3P and provides molecular evidence for the importance of this lipid in the vesicular trafficking pathway of host-cell cytosol, and apicoplast and mitochondrion dynamics.
Hepatitis E virus (HEV) infection is a major cause of acute viral hepatitis worldwide. HEV is a positive-strand RNA virus encoding three open reading frames (ORFs). The ORF3 protein is a small membrane-associated protein essential for viral particle secretion; however, its precise role in the viral life cycle remains incompletely understood. Here, we performed immunoprecipitation followed by mass spectrometry to identify host proteins interacting with the HEV ORF3 protein. Candidate interactors were validated by co-immunoprecipitation, confirming physical interactions between ORF3 and cysteine-rich and transmembrane domain-containing protein 1 (CYSTM1), Ras-related protein Rab24, and transferrin receptor 1 (TfR1). Confocal microscopy demonstrated colocalization of all three host factors with ORF3 protein. Gene silencing and knockout revealed that each protein contributes to virus production, with TfR1 depletion producing the most pronounced effect. In cells harboring replicating HEV, TfR1 colocalized with the ORF2 (capsid) and ORF3 proteins at Rab11A-positive recycling endosomes. Silencing of TfR1 in primary human hepatocytes, followed by HEV RNA transfection or infection, confirmed its role in virus production, particularly in the assembly of infectious particles, consistent with its colocalization with the HEV ORF2 protein. Collectively, our proteomics-based analysis identifies TfR1, along with CYSTM1 and Rab24, as novel ORF3-interacting host factors required for efficient production of infectious HEV. These findings provide new insights into the role of ORF3 protein in viral assembly and highlight TfR1 as a key host factor in the HEV life cycle.
Prions are misfolded, self-propagating versions of cellular proteins. In humans and animals, misfolding of the cellular prion protein (PrP C ) causes invariably fatal transmissible neurodegenerative diseases. Little is known about how mammalian prions replicate in the brain, including whether other proteins participate in prion replication in vivo . Several members of the protein disulfide isomerase family have been shown to reside in close spatial proximity to PrP C in cells and mice, implying that they could be involved in prion biogenesis. Here, we show that stable knock-down of the protein disulfide isomerase P4HB (also called PDIA1) in prion-susceptible CAD5 cells reduces PrP C levels and hinders the generation of protease-resistant PrP (PrP res ) following infection with two different prion strains. Moreover, transient knock-down of P4HB decreases PrP res levels in cells with established prion infection. Partial reduction of P4HB activity using the P4HB-selective inhibitor KSC-34 also decreases PrP C levels in uninfected CAD5 cells whereas treatment of prion-infected CAD5 cells with KSC-34 results in higher levels of PrP res . A proportion of P4HB reaches the cell surface where PrP C is located, and a secreted P4HB variant increases PrP res levels in cells. Collectively, these results suggest that P4HB influences PrP C homeostasis and modulates the conversion of PrP C into misfolded species. Thus, targeting P4HB during prion disease may have therapeutic benefit.
The cell surface of bloodstream-form African trypanosomes is covered by a dense coat of variant surface glycoproteins (VSGs). By periodically switching the expressed VSG antigen, parasites evade host immune responses. VSG mRNA constitutes ~10% of total cellular mRNA, and depletion of VSG transcripts is lethal. When two VSGs are expressed simultaneously, however, total VSG mRNA levels remain close to wild-type amounts, indicating the presence of a balancing mechanism that limits the overall abundance of these highly expressed transcripts. Using inducible and constitutive expression systems, we found that attenuation of endogenous VSG mRNA requires efficient engagement of ectopic transcripts with the endoplasmic reticulum (ER). This response occurs independently of efficient VSG protein production and does not require a VSG open reading frame. In contrast, abundant transcripts lacking functional ER-engagement signals fail to trigger balancing despite containing the VSG 3' UTR 16-mer stability element. These results indicate that the signal for VSG mRNA regulation is the presence of abundant ER-engaged transcripts (i.e., transcripts undergoing co-translational targeting to the ER), rather than VSG-specific sequence features or productive protein synthesis. Based on our findings together with previous work, we propose an ER-engagement-coupled homeostatic attenuation mechanism in which increased ER-engaged transcript load elicits a regulatory response that reduces endogenous VSG mRNA abundance. This model links the cytoplasmic burden of ER-targeted transcripts to transcriptional control of the nuclear VSG expression site, providing a mechanism that could allow trypanosomes to couple secretory pathway capacity to surface antigen expression. Such a mechanism could enable trypanosomes to maintain secretory pathway homeostasis while supporting rapid surface-coat remodelling during antigenic variation.
We previously showed that two natural, single-amino acid mutations in the HCPro of a potyviral chimera, separately increased virulence by enhancing either its silencing-suppression or proteolytic functions. To investigate how beneficial variants generated during infection compete with non-mutant forms, or among each other, we co-expressed them in pairs, in Nicotiana benthamiana . We assessed how variants co-exist and influence each other in the cell, and whether altered abiotic (elevated temperature) or biotic (a silencing-impaired plant) environmental factors, affected local and systemic infection outcomes. In local tissues, our results are consistent with a model in which variants replicate and accumulate independently in co-infected cells: observed variant titers and ratios correlated with those that could be deduced from single infections. Trans-acting effects of one variant over the other were not observed. By contrast, environment parameters influenced not only local titers but also ratios, likely because of their differential effects on functions affected by the specific mutation of each variant. Additional mutations in HCPro appeared separately in systemic infections in specific plants, and induced either new substitutions in the protein, or in a putative small reverse reading frame. Thus, further adaptation could proceed through separate paths via the incorporation of distinct, novel mutations in the HCPro sequence in different plants.
Crimean-Congo hemorrhagic fever (CCHF), which is caused by infection with the CCHF virus (CCHFV), is the most widespread hemorrhagic infectious disease. In severe cases, liver damage is a salient manifestation. However, the detailed mechanism is not yet fully understood. To investigate the pathogenesis of CCHF-related liver damage, we infected type I interferon receptor 1 knockout (IFNAR1 -/- ) mice with Hazara virus (HAZV), which is a surrogate pathogen of CCHFV, as well as with the CCHFV itself. HAZV infection caused CCHF-like symptoms, including severe liver damage, alongside inflammatory responses. HAZV infection in IFNAR1 -/- mice additionally lacking mitochondrial antiviral signaling protein (MAVS) induced minimal cytokine responses; however, these mice still exhibited weight loss and liver damage, albeit with a significantly delayed onset of lethal outcomes. We found that loss of liver-resident macrophage, Kupffer cells, occurred prior to viral spread in hepatocytes and liver damage in these mice. Notably, mice lacking IFNAR1 in Kupffer cells, but not mice lacking IFNAR1 in hepatocytes, also lost Kupffer cell population and exhibited lethal liver damage, following HAZV and CCHFV infection. Our findings indicate that IFNAR1 signaling in mononuclear phagocytes, especially Kupffer cells, is essential for preventing viral spread and fatal liver damage and raise the possibility that inflammatory cytokines- and viral replication-mediated Kupffer cell loss synergistically drives both processes.
