PLoS Pathogens最新文献

Toscana virus is a major cause of arboviral disease in humans in the Mediterranean basin during summer. However, early virus-host cell interactions and entry mechanisms remain poorly characterized. Investigating iPSC-derived human neurons and cell lines, we found that virus binding to the cell surface was specific, and 50% of bound virions were endocytosed within 10 min. Virions entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomal compartments. Penetration required intact late endosomes and occurred within 30 min following internalization. Virus entry relied on vacuolar acidification, with an optimal pH for viral membrane fusion at pH 5.5. The pH threshold increased to 5.8 with longer pre-exposure of virions to the slightly acidic pH in early endosomes. Strikingly, the particles remained infectious after entering late endosomes with a pH below the fusion threshold. Overall, our study establishes Toscana virus as a late-penetrating virus and reveals an atypical use of vacuolar acidity by this virus to enter host cells.

Pathogenic Xanthomonas bacteria cause disease on more than 400 plant species. These Gram-negative bacteria utilize the type III secretion system to inject type III effector proteins (T3Es) directly into the plant cell cytosol where they can manipulate plant pathways to promote virulence. The host range of a given Xanthomonas species is limited, and T3E repertoires are specialized during interactions with specific plant species. Some effectors, however, are retained across most strains, such as Xanthomonas Outer Protein L (XopL). As an 'ancestral' effector, XopL contributes to the virulence of multiple xanthomonads, infecting diverse plant species. XopL homologs harbor a combination of a leucine-rich-repeat (LRR) domain and an XL-box which has E3 ligase activity. Despite similar domain structure there is evidence to suggest that XopL function has diverged, exemplified by the finding that XopLs expressed in plants often display bacterial species-dependent differences in their sub-cellular localization and plant cell death reactions. We found that XopL from X. euvesicatoria (XopLXe) directly associates with plant microtubules (MTs) and causes strong cell death in agroinfection assays in N. benthamiana. Localization of XopLXe homologs from three additional Xanthomonas species, of diverse infection strategy and plant host, revealed that the distantly related X. campestris pv. campestris harbors a XopL (XopLXcc) that fails to localize to MTs and to cause plant cell death. Comparative sequence analyses of MT-binding XopLs and XopLXcc identified a proline-rich-region (PRR)/α-helical region important for MT localization. Functional analyses of XopLXe truncations and amino acid exchanges within the PRR suggest that MT-localized XopL activity is required for plant cell death reactions. This study exemplifies how the study of a T3E within the context of a genus rather than a single species can shed light on how effector localization is linked to biochemical activity.

In this study, we evaluated the impact of viral variant, in addition to other variables, on within-host viral burden, by analysing cycle threshold (Ct) values derived from nose and throat swabs, collected as part of the UK COVID-19 Infection Survey. Because viral burden distributions determined from community survey data can be biased due to the impact of variant epidemiology on the time-since-infection of samples, we developed a method to explicitly adjust observed Ct value distributions to account for the expected bias. By analysing the adjusted Ct values using partial least squares regression, we found that among unvaccinated individuals with no known prior exposure, viral burden was 44% lower among Alpha variant infections, compared to those with the predecessor strain, B.1.177. Vaccination reduced viral burden by 67%, and among vaccinated individuals, viral burden was 286% higher among Delta variant, compared to Alpha variant, infections. In addition, viral burden increased by 17% for every 10-year age increment of the infected individual. In summary, within-host viral burden increases with age, is reduced by vaccination, and is influenced by the interplay of vaccination status and viral variant.

The multigene family genes (MGFs) in the left variable region (LVR) of the African swine fever virus (ASFV) genome have been reported to be involved in viral replication in primary porcine alveolar macrophages (PAMs) and virulence in pigs. However, the exact functions of key MGFs in the LVR that regulate the replication and virulence of ASFV remain unclear. In this study, we identified the MGF300-2R gene to be critical for viral replication in PAMs by deleting different sets of MGFs in the LVR from the highly virulent strain ASFV HLJ/18 (ASFV-WT). The ASFV mutant lacking the MGF300-2R gene (Del2R) showed a 1-log reduction in viral titer, and induced higher IL-1β and TNF-α production in PAMs than did ASFV-WT. Mechanistically, the MGF300-2R protein was found to interact with and degrade IKKα and IKKβ via the selective autophagy pathway. Furthermore, we showed that MGF300-2R promoted the K27-linked polyubiquitination of IKKα and IKKβ, which subsequently served as a recognition signal for the cargo receptor TOLLIP-mediated selective autophagic degradation. Importantly, Del2R exhibited a significant reduction in both replication and virulence compared with ASFV-WT in pigs, likely due to the increased IL-1β and TNF-α, indicating that MGF300-2R is a virulence determinant. These findings reveal that MGF300-2R suppresses host innate immune responses by mediating the degradation of IKKα and IKKβ, which provides clues to paving the way for the rational design of live attenuated vaccines to control ASF.

Innate immune priming increases an organism's survival of a second infection after an initial, non-lethal infection. We used Drosophila melanogaster and an insect-derived strain of Enterococcus faecalis to study transcriptional control of priming. In contrast to other pathogens, the enhanced survival in primed animals does not correlate with decreased E. faecalis load. Further analysis shows that primed organisms tolerate, rather than resist infection. Using RNA-seq of immune tissues, we found many genes were upregulated in only primed flies, suggesting a distinct transcriptional program in response to initial and secondary infections. In contrast, few genes continuously express throughout the experiment or more efficiently re-activate upon reinfection. Priming experiments in immune deficient mutants revealed Imd is largely dispensable for responding to a single infection but needed to fully prime. Together, this indicates the fly's innate immune response is plastic-differing in immune strategy, transcriptional program, and pathway use depending on infection history.

The SARS-CoV-2 pandemic has led to the emergence of various variants of concern (VoCs) that are associated with increased transmissibility, immune evasion, or differences in disease severity. The emergence of VoCs fueled interest in understanding the potential impact of travel restrictions and surveillance strategies to prevent or delay the early spread of VoCs. We performed phylogenetic analyses and mathematical modeling to study the importation and spread of the VoCs Alpha and Delta in Switzerland in 2020 and 2021. Using a phylogenetic approach, we estimated between 383-1,038 imports of Alpha and 455-1,347 imports of Delta into Switzerland. We then used the results from the phylogenetic analysis to parameterize a dynamic transmission model that accurately described the subsequent spread of Alpha and Delta. We modeled different counterfactual intervention scenarios to quantify the potential impact of border closures and surveillance of travelers on the spread of Alpha and Delta. We found that implementing border closures after the announcement of VoCs would have been of limited impact to mitigate the spread of VoCs. In contrast, increased surveillance of travelers could prove to be an effective measure for delaying the spread of VoCs in situations where their severity remains unclear. Our study shows how phylogenetic analysis in combination with dynamic transmission models can be used to estimate the number of imported SARS-CoV-2 variants and the potential impact of different intervention scenarios to inform the public health response during the pandemic.

Mucins play an essential role in protecting the respiratory tract against microbial infections while also acting as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance. Transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on epithelial entry of SARS-CoV-2. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of terminal mucin glycans sialic acid and fucose from the epithelial surface did not impact viral entry. In Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are located to the apical surface in close proximity and StcE treatment results in enhanced binding of purified spike protein. Both MUC1 and MUC16 are expressed on the surface of human organoid-derived air-liquid interface (ALI) differentiated airway cultures and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 replication. In these cultures, MUC1 was highly expressed in non-ciliated cells while MUC16 was enriched in goblet cells. In conclusion, the glycosylated extracellular domains of different transmembrane mucins might have similar protective functions in different respiratory cell types by restricting SARS-CoV-2 binding and entry.

Fungal insect pathogens have evolved diverse mechanisms to evade host immune recognition and defense responses. However, identification of fungal factors involved in host immune evasion during cuticular penetration and subsequent hemocoel colonization remains limited. Here, we report that the entomopathogenic fungus Beauveria bassiana expresses an endo-β-1,3-glucanase (BbEng1) that functions in helping cells evade insect immune recognition/ responses. BbEng1 was specifically expressed during infection, in response to host cuticle and hemolymph, and in the presence of osmotic or oxidative stress. BbEng1 was localized to the fungal cell surface/ cell wall, where it acts to remodel the cell wall pathogen associated molecular patterns (PAMPs) that can trigger host defenses, thus facilitating fungal cell evasion of host immune defenses. BbEng1 was secreted where it could bind to fungal cells. Cell wall β-1,3-glucan levels were unchanged in ΔBbEng1 cells derived from in vitro growth media, but was elevated in hyphal bodies, whereas glucan levels were reduced in most cell types derived from the BbEng1 overexpressing strain (BbEng1OE). The BbEng1OE strain proliferated more rapidly in the host hemocoel and displayed higher virulence as compared to the wild type parent. Overexpression of their respective Eng1 homologs or of BbEng1 in the insect fungal pathogens, Metarhizium robertsii and M. acridum also resulted in increased virulence. Our data support a mechanism by which BbEng1 helps the fungal pathogen to evade host immune surveillance by decreasing cell wall glucan PAMPs, promoting successful fungal mycosis.

Zika virus (ZIKV) can be transmitted vertically from mother to fetus during pregnancy, resulting in a range of outcomes including severe birth defects and fetal/infant death. Potential pathways of vertical transmission in utero have been proposed but remain undefined. Identifying the timing and routes of vertical transmission of ZIKV may help us identify when interventions would be most effective. Furthermore, understanding what barriers ZIKV overcomes to effect vertical transmission may help improve models for evaluating infection by other pathogens during pregnancy. To determine the pathways of vertical transmission, we inoculated 12 pregnant rhesus macaques with an African-lineage ZIKV at gestational day 30 (term is 165 days). Eight pregnancies were surgically terminated at either seven or 14 days post-maternal infection. Maternal-fetal interface and fetal tissues and fluids were collected and evaluated for ZIKV using RT-qPCR, in situ hybridization, immunohistochemistry, and plaque assays. Four additional pregnant macaques were inoculated and terminally perfused with 4% paraformaldehyde at three, six, nine, or ten days post-maternal inoculation. For these four cases, the entire fixed pregnant uterus was evaluated with in situ hybridization for ZIKV RNA. We determined that ZIKV can reach the MFI by six days after infection and infect the fetus by ten days. Infection of the chorionic membrane and the extraembryonic coelomic fluid preceded infection of the fetus and the mesenchymal tissue of the placental villi. We did not find evidence to support a transplacental route of ZIKV vertical transmission via infection of syncytiotrophoblasts or villous cytotrophoblasts. The pattern of infection observed in the maternal-fetal interface provides evidence of paraplacental vertical ZIKV transmission through the chorionic membrane, the outer layer of the fetal membranes.

The genetic causes of phenotypic variation often differ depending on the population examined, particularly if the populations were founded by relatively small numbers of genotypes. Similarly, the genetic causes of phenotypic variation among similar traits (resistance to different xenobiotic compounds or pathogens) may also be completely different or only partially overlapping. Differences in genetic causes for variation in the same trait among populations suggests context dependence for how selection acts on those traits. Similarities in the genetic causes of variation for different traits, on the other hand, suggests pleiotropy which would also influence how natural selection shapes variation in a trait. We characterized immune defense against a natural Drosophila pathogen, the Gram-positive bacterium Lysinibacillus fusiformis, in three different populations and found almost no overlap in the genetic architecture of variation in survival post infection. However, when comparing our results to a similar experiment with the fungal pathogen, B. bassiana, we found a convincing shared QTL peak for both pathogens. This peak contains the Bomanin cluster of Drosophila immune effectors. Loss of function mutants and RNAi knockdown experiments confirms a role of some of these genes in immune defense against both pathogens. This suggests that natural selection may act on the entire cluster of Bomanin genes (and the linked region under the QTL) or specific peptides for specific pathogens.