PLoS Pathogens最新文献

The Helicobacter pylori flagellar motor contains several accessory structures that are not found in the archetypal Escherichia coli and Salmonella enterica motors. H. pylori hp0838 encodes a previously uncharacterized lipoprotein and is in an operon with flgP, which encodes a motor accessory protein. Deletion analysis of hp0838 in H. pylori B128 showed that the gene is not required for motility in soft agar medium, but the mutant displayed a reduced growth rate and an increased sensitivity to bacitracin, which is an antibiotic that is normally excluded by the outer membrane. Introducing a plasmid-borne copy of hp0838 into the H. pylori Δhp0838 mutant suppressed the fitness defect and antibiotic sensitivity of the strain. A variant of the Δhp0838 mutant containing a frameshift mutation in pflA, which resulted in paralyzed flagella, displayed wild-type growth rate and resistance to bacitracin, suggesting the fitness defect and antibiotic sensitivity of the Δhp0838 mutant are dependent on flagellar rotation. Comparative analysis of in-situ structures of the wild type and Δhp0838 mutant motors revealed the Δhp0838 mutant motor lacked a previously undescribed ring structure with 18-fold symmetry located near the outer membrane. Given its role in formation of the motor outer ring, HP0838 was designated FapH (flagellar accessory protein in Helicobacter pylori) and the motor accessory formed the protein was named the FapH ring. Our data suggest that the FapH ring helps to preserve outer membrane barrier function during flagellar rotation. Given that FapH homologs are present in many members of the phylum Campylobacterota, they may have similar roles in protecting the outer membrane from damage due to flagellar rotation in these bacteria.

Host-pathogen interactions represent a dynamic evolutionary process, wherein both hosts and pathogens continuously develop complex mechanisms to outmaneuver each other. Borrelia burgdorferi, the Lyme disease pathogen, has evolved an intricate antigenic variation mechanism to evade the host immune response, enabling its dissemination, persistence, and pathogenicity. Despite the discovery of this mechanism over two decades ago, the precise processes, genetic elements, and proteins involved in this system remain largely unknown. The vls locus, which is the site of antigenic variation, has been notoriously challenging to manipulate genetically due to its highly conserved structural features, even with significant advancements in molecular biology and genetic engineering for this highly segmented pathogen. Our study highlights the pivotal role of plasmid topology in facilitating in trans gene recombination. We demonstrate that gene conversion can occur in trans when a copy of vlsE gene is present on a linear plasmid, contrary to previous observations suggesting a cis arrangement is required for vlsE recombination. Significantly, employing this in trans gene conversion strategy with a linear plasmid, we have, for the first time, achieved targeted genetic mutation of putative cis-acting elements in the native vlsE gene. This has unveiled a potentially crucial role for the 17 bp direct repeats that flank the central variable cassette region of vlsE. Furthermore, we validated the reliability and reproducibility of our mutational approach by successfully inserting stop codons at two distinct sites within the central variable cassette of vlsE. Thus, this study presents a significant methodological innovation enabling the direct manipulation of the vls locus and lays the groundwork for systematic exploration of specific mutations affecting the mechanism of antigenic variation. As a result, it creates new avenues for research and raises intriguing questions that could guide the development of novel methods to explore host-pathogen interactions of the agent of Lyme disease.

HBV genotype A has two major subtypes, A1 (commonly in Africa) and A2 (commonly in Europe) with only 4% nucleotide differences. Individuals infected with these two subtypes appear to have different clinical manifestations and virologic features. Whether such a difference results from the virus or host has not been established. Using HBV generated from molecule clones of subtypes A1 and A2 in cell culture (HBVcc), we demonstrate that HBVcc of subtypes A1 and A2 can be passaged in vitro and in vivo and respond equally well to human IFN-α treatment. HBVcc passaged in human liver chimeric mice (HBVmp) infected human hepatocytes more efficiently than that of the original HBVcc. Subtype A2 showed a much higher viral replication level than that of subtype A1. Mechanistic investigations using constructs with chimeric A1/A2 sequences and specific mutations indicated that subtype A2 has an inherently higher replication phenotype due to specific polymorphisms in the HBx gene resulting in amino acid variations. Studies of HBx expression demonstrated that A1 HBx is expressed at a much lower level than that of A2 HBx. Mutagenesis studies identified two HBx amino acid variations responsible for the observed phenotypic difference. Using AlphaFold2, we generated structural models of HBx proteins of A1 and A2. Superposition of the two models reveal that the overall structural motifs are similarly aligned, except for the C-terminal peptides diverging between the A1 and A2 models, possibly explaining their functional difference. In conclusion, using various in vitro and in vivo models, here we show that subtype A2 has an inherently higher replication phenotype due to polymorphisms in HBx that result in possible differences in structure and expression level of the two subtype HBx proteins. This genotypic difference potentially explains the reported clinical differences between the two subtypes as well as providing a previously unrecognized association between viral sequence variations and clinical manifestations of HBV infection in humans.

Filamentous plant pathogenic fungi pose significant threats to global food security, particularly through diseases like Fusarium Head Blight (FHB) and Septoria Tritici Blotch (STB) which affects cereals. With mounting challenges in fungal control and increasing restrictions on fungicide use due to environmental concerns, there is an urgent need for innovative control strategies. Here, we present a comprehensive analysis of the stage-specific infection process of Fusarium graminearum in wheat spikes by generating a dual weighted gene co-expression network (WGCN). Notably, the network contained a mycotoxin-enriched fungal module (F12) that exhibited a significant correlation with a detoxification gene-enriched wheat module (W12). This correlation in gene expression was validated through quantitative PCR. By examining a fungal module with genes highly expressed during early symptomless infection that was correlated to a wheat module enriched in oxidative stress genes, we identified a gene encoding FgKnr4, a protein containing a Knr4/Smi1 disordered domain. Through comprehensive analysis, we confirmed the pivotal role of FgKnr4 in various biological processes, including oxidative stress tolerance, cell cycle stress tolerance, morphogenesis, growth, and pathogenicity. Further studies confirmed the observed phenotypes are partially due to the involvement of FgKnr4 in regulating the fungal cell wall integrity pathway by modulating the phosphorylation of the MAP-kinase MGV1. Orthologues of the FgKnr4 gene are widespread across the fungal kingdom but are absent in other Eukaryotes, suggesting the protein has potential as a promising intervention target. Encouragingly, the restricted growth and highly reduced virulence phenotypes observed for ΔFgknr4 were replicated upon deletion of the orthologous gene in the wheat fungal pathogen Zymoseptoria tritici. Overall, this study demonstrates the utility of an integrated network-level analytical approach to pinpoint genes of high interest to pathogenesis and disease control.

Vaccines are widely regarded as one of the most effective strategies for combating infectious diseases. However, significant challenges remain, such as insufficient antibody levels, limited protection against rapidly evolving variants, and poor immune durability, particularly in subunit vaccines, likely due to their short in vivo exposure. Recent advances in extending the half-life of protein therapeutics have shown promise in improving drug efficacy, yet whether increasing in vivo persistence can enhance the efficacy of subunit vaccines remains underexplored. In this study, we developed two trimeric SARS-CoV-2 subunit vaccines with distinct pharmacokinetic profiles to evaluate the impact of vaccine persistence on immune efficacy. A self-assembling trimeric subunit vaccine (RBD-HR/trimer) was designed, followed by an extended-persistence variant (RBD-sFc-HR/trimer) incorporating a soluble monomeric IgG1 fragment crystallizable. We demonstrated that RBD-sFc-HR/trimer elicited more robust and higher levels of neutralizing antibodies, with potent and broad neutralization activity against multiple SARS-CoV-2 variants. Notably, RBD-sFc-HR/trimer induced a durable immune response, significantly increasing the number of memory B cells and T cells. This study provides critical insights for designing vaccines that achieve potent and long-lasting immune responses against infectious diseases.

Long non-coding RNAs (lncRNAs) are essential components of innate immunity, maintaining the functionality of immune systems that control virus infection. However, how lncRNAs engage immune responses during influenza A virus (IAV) infection remains unclear. Here, we show that lncRNA USP30-AS1 is up-regulated by infection of multiple different IAV subtypes and is required for tuning inflammatory and antiviral response in IAV infection. Genetically inactivation of USP30-AS1 enhances viral protein synthesis and viral growth. USP30-AS1 is an interferon-stimulated gene, and the induction of USP30-AS1 can be achieved by JAK-STAT mediated signaling activation. The immune regulation of USP30-AS1 is independent of its proximal protein-coding gene USP30. In IAV infection, deletion of USP30-AS1 unleashes high systemic inflammatory responses involving a broad range of pro-inflammatory factors, suggesting USP30-AS1 as a critical modulator of immune responses in IAV infection. Furthermore, we established a database providing well-annotated host gene expression profiles IAV infection or immune stimulation.

Spirochetes are a widely existing group of bacteria with a distinct morphology. Some spirochetes are important human pathogens that utilize periplasmic flagella to achieve motility and host infection. The motors that drive the rotation of periplasmic flagella have a unique spirochete-specific feature, termed the collar, crucial for the flat-wave morphology and motility of the Lyme disease spirochete Borrelia burgdorferi. Here, we deploy cryo-electron tomography and subtomogram averaging to determine high-resolution in-situ structures of the B. burgdorferi flagellar motor. Comparative analysis and molecular modeling of in-situ flagellar motor structures from B. burgdorferi mutants lacking each of the known collar proteins (FlcA, FlcB, FlcC, FlbB, and Bb0236/FlcD) uncover a complex protein network at the base of the collar. Importantly, our data suggest that FlbB not only forms a novel periplasmic ring around the rotor but also acts as a scaffold supporting collar assembly and subsequent recruitment of stator complexes. The complex protein network based on the FlbB ring effectively bridges the rotor and 16 torque-generating stator complexes in each flagellar motor, thus contributing to the specialized motility and lifestyle of spirochetes in complex environments.

Malaria parasites must respond quickly to environmental changes, including during their transmission between mammalian and mosquito hosts. Therefore, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. While the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, changes in the spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii, we found that ~200 transcripts are released for translation soon after fertilization, including those encoding essential functions. Moreover, we identified that many transcripts remain repressed beyond this point. TurboID-based proximity proteomics of the DOZI/CITH/ALBA regulatory complex revealed substantial spatial and/or compositional changes across this transmission event, which are consistent with recent, paradigm-shifting models of translational control. Together, these data provide a model for the essential translational control mechanisms that promote Plasmodium's efficient transmission from mammalian host to mosquito vector.

Japanese encephalitis virus (JEV), a neuroinvasive and neurovirulent orthoflavivirus, can be prevented in humans with the SA14-14-2 vaccine, a live-attenuated version derived from the wild-type SA14 strain. To determine the viral factors responsible for the differences in pathogenicity between SA14 and SA14-14-2, we initially established a reverse genetics system that includes a pair of full-length infectious cDNAs for both strains. Using this cDNA pair, we then systematically exchanged genomic regions between SA14 and SA14-14-2 to generate 20 chimeric viruses and evaluated their replication capability in cell culture and their pathogenic potential in mice. Our findings revealed the following: (i) The single envelope (E) protein of SA14-14-2, which contains nine mutations (eight in the ectodomain and one in the stem region), is both necessary and sufficient to render SA14 non-neuroinvasive and non-neurovirulent. (ii) Conversely, the E protein of SA14 alone is necessary for SA14-14-2 to become highly neurovirulent, but it is not sufficient to make it highly neuroinvasive. (iii) The limited neuroinvasiveness of an SA14-14-2 derivative that contains the E gene of SA14 significantly increases (approaching that of the wild-type strain) when two viral nonstructural proteins are replaced by their counterparts from SA14: (a) NS1/1', which has four mutations on the external surface of the core β-ladder domain; and (b) NS2A, which has two mutations in the N-terminal region, including two non-transmembrane α-helices. In line with their roles in viral pathogenicity, the E, NS1/1', and NS2A genes all contribute to the enhanced spread of the virus in cell culture. Collectively, our data reveal for the first time that the E protein of JEV has a dual function: It is the master regulator of viral neurovirulence and also the primary initiator of viral neuroinvasion. After the initial E-mediated neuroinvasion, the NS1/1' and NS2A proteins act as secondary promoters, further amplifying viral neuroinvasiveness.

Microsporidia are a group of intracellular pathogens that actively manipulate host cell biological processes to facilitate their intracellular niche. Apoptosis is an important defense mechanism by which host cell control intracellular pathogens. Microsporidia modulating host cell apoptosis has been reported previously, however the molecular mechanism is not yet clear. In this report, we describe that the microsporidia Nosema bombycis inhibits apoptosis of Bombyx mori cells through a secreted protein NbSPN14, which is a serine protease inhibitor (Serpin). An immunofluorescent assay demonstrated that upon infection with N. bombycis, NbSPN14 was initially found in the B. mori cell cytoplasm and then became enriched in the host cell nucleus. Overexpression and RNA-interference (RNAi) of NbSPN14 in B. mori' embryo cell confirmed that NbSPN14 inhibited host cells apoptosis. Immunofluorescent and Co-IP assays verified the co-localization and interaction of NbSPN14 with the BmICE, the Caspase 3 homolog in B. mori. Knocking out of BmICE or mutating the BmICE-interacting P1 site of NbSPN14, eliminated the localization of NbSPN14 into the host nucleus and prevented the apoptosis-inhibiting effect of NbSPN14, which also proved that the interaction between BmICE and NbSPN14 occurred in host cytoplasm and the NbSPN14 translocation into host cell nucleus is depends on BmICE. These data elucidate that N. bombycis secretory protein NbSPN14 inhibits host cell apoptosis by directly inhibiting the Caspase protease BmICE, which provides an important insight for understanding pathogen-host interactions and a potential therapeutic target for N. bombycis proliferation.