PLoS Pathogens最新文献

Porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis coronavirus (TGEV), the two causative agents of porcine diarrhea, have been reported to be at risk of cross-species transmission, including to humans. However, the potential host range in which these two CoVs interact remains unclear. We screened 16 animal counterparts for porcine aminopeptidase N (APN), the receptor of PDCoV and TGEV, and found that APNs from eight of 17 animals could bind to the receptor-binding domains (RBDs) of PDCoV and TGEV. Furthermore, the animal APNs that could bind to the RBDs could mediate cellular infection by both viruses. Dog APN (dAPN) has been identified as the animal receptor with the highest capability to mediate the virus infection. We further resolved the complex structures of dAPN bound to the PDCoV RBD/TGEV RBD, respectively, establishing its divergent receptor-binding modes. We identified R325 of dAPN as an important residue in the PDCoV RBD-dAPN interaction, and found the central role of Q746 and T749 in dAPN in the interaction with the TGEV RBD. These findings provide the molecular basis of the potential cross-species transmission of these two porcine CoVs and shed light on future surveillance of these CoVs.

Compromised male reproductive health, including reduced testosterone and sperm count, is one of the long COVID symptoms in individuals recovering from mild-severe disease. COVID-19 patients display testicular injury in the acute stage and altered serum fertility markers in the recovery phase, however, long-term implications on the testis remain unknown. This study characterized the consequences of SARS-CoV-2 on testis function. The K18-hACE2 mice that survived SARS-CoV-2 infection were followed for one month after infection and the testicular injury and function markers were assessed at different stages of infection and recovery. The long-term impact of infection on key testes function-related hormones and male fertility was measured. The efficacy of inflammation-suppressing drug in preventing testicular injury was also evaluated. The morphological defects like sloughing of spermatids into the lumen and increased apoptotic cells sustained for 2-4 weeks after infection and correlated with testicular inflammation and immune cell infiltration. Transcriptomic analysis revealed dysregulation of inflammatory, cell death, and steroidogenic pathways. Furthermore, reduced testosterone levels associated with a transient reduction in sperm count and male fertility. Most testicular impairments resolved within one month of infection. Importantly, dexamethasone treatment attenuated testicular damage, inflammation, and immune infiltration. Our results implicate virus-induced cytokine storm as the major driver of testicular injury and functional impairments, timely prevention of which limits testis damage. These findings serve as a model for evaluating therapeutics in long COVID patients and may guide clinical strategies to improve male reproductive health outcomes post-SARS-CoV-2 infection.

Wheat production is threatened by multiple fungal pathogens, such as the wheat powdery mildew fungus (Blumeria graminis f. sp. tritici, Bgt). Wheat resistance breeding frequently relies on the use of resistance (R) genes that encode diverse immune receptors which detect specific avirulence (AVR) effectors and subsequently induce an immune response. While R gene cloning has accelerated recently, AVR identification in many pathogens including Bgt lags behind, preventing pathogen-informed deployment of resistance sources. Here we describe a new "avirulence depletion (AD) assay" for rapid identification of AVR genes in Bgt. This assay relies on the selection of a segregating, haploid F1 progeny population on a resistant host, followed by bulk sequencing, thereby allowing rapid avirulence candidate gene identification with high mapping resolution. In a proof-of-concept experiment we mapped the AVR component of the wheat immune receptor Pm3a to a 25 kb genomic interval in Bgt harboring a single effector, the previously described AvrPm3a2/f2. Subsequently, we applied the AD assay to map the unknown AVR effector recognized by the Pm60 immune receptor. We show that AvrPm60 is encoded by three tandemly arrayed, nearly identical effector genes that trigger an immune response upon co-expression with Pm60 and its alleles Pm60a and Pm60b. We furthermore provide evidence that Pm60 outperforms Pm60a and Pm60b through more efficient recognition of AvrPm60 effectors, suggesting it should be prioritized for wheat breeding. Finally, we show that virulence towards Pm60 is caused by simultaneous deletion of all AvrPm60 gene paralogs and that isolates lacking AvrPm60 are especially prevalent in the US thereby limiting the potential of Pm60 in this region. The AD assay is a powerful new tool for rapid and inexpensive AVR identification in Bgt with the potential to contribute to pathogen-informed breeding decisions for the use of novel R genes and regionally tailored gene deployment.

Chronic hepatitis B virus (HBV) infection can significantly increase the incidence of cirrhosis and liver cancer, and there is no curative treatment. The persistence of HBV covalently closed circular DNA (cccDNA) is the major obstacle of antiviral treatments. cccDNA is formed through repairing viral partially double-stranded relaxed circular DNA (rcDNA) by varies host factors. However, the detailed mechanisms are not well characterized. To dissect the biogenesis of cccDNA, we took advantage of an in vitro rcDNA repair system to precipitate host factors interacting with rcDNA and identified co-precipitated proteins by mass spectrometry. Results revealed the MRE11-RAD50-NBS1 (MRN) complex as a potential factor. Transiently or stably knockdown of MRE11, RAD50 or NBS1 in hepatocytes before HBV infection significantly decreased viral markers, including cccDNA, while reconstitution reversed the effect. Chromatin immunoprecipitation assay further validated the interaction of MRN complex and HBV DNA. However, MRN knockdown after HBV infection showed no effect on viral replication, which indicated that MRN complex inhibited the formation of cccDNA without affecting its stability or transcriptional activity. Interestingly, Mirin, a MRN complex inhibitor which can inhibit the exonuclease activity of MRE11 and MRN-dependent activation of ATM, but not ATM kinase inhibitor KU55933, could decrease cccDNA level. Likewise, the MRE11 endonuclease activity inhibitor PFM01 treatment decreased cccDNA. MRE11 nuclease assays indicated that rcDNA is a substrate of MRE11. Furthermore, the inhibition of ATR-CHK1 pathway, which is known to be involved in cccDNA formation, impaired the effect of MRN complex on cccDNA. Similarly, inhibition of MRE11 endonuclease activity mitigated the effect of ATR-CHK1 pathway on cccDNA. These findings indicate that MRN complex cooperates with ATR-CHK1 pathway to regulate the formation of HBV cccDNA. In summary, we identified host factors, specifically the MRN complex, regulating cccDNA formation during HBV infection. These findings provide insights into how HBV hijacks host enzymes to establish chronic infection and reveal new therapeutic opportunities.

Virus-derived small interfering RNAs (vsiRNAs) have been widely recognized to play an antiviral immunity role. However, it is unclear whether vsiRNAs can also play a positive role in viral infection. Here, we characterized three highly abundant vsiRNAs mapped to the genomic termini of rice stripe virus (RSV), a negative-strand RNA virus transmitted by insect vectors. The three vsiRNAs shared 11 nucleotides due to the conservative genomic termini and were likely generated from viral terminal panhandle structure, depending on both Dicer1 and Dicer2 in insects. In addition to targeting viral RNAs in a miRNA-like manner, the three vsiRNAs coordinately downregulated the expression of DOPA decarboxylase, thereby suppressing the prophenoloxidase immune reaction in insect vectors. In vsiRNA-silenced transgenic rice, the viral titer significantly decreased, indicating that these vsiRNAs promote RSV replication in rice. This study elucidates a unique function of vsiRNAs derived from the conserved panhandle structure of negative-strand RNA viruses in enhancing viral infection.

Sodium taurocholate co-transporting polypeptide (NTCP) has been identified as an entry receptor for hepatitis B virus (HBV), but the molecular events of the viral post-endocytosis steps remain obscure. In this study, we discovered that manganese (Mn) could strongly inhibit HBV infection in NTCP-reconstituted HepG2 cells without affecting viral replication. We therefore profiled the antiviral effects of Mn2+ in an attempt to elucidate the regulatory mechanisms involved in early HBV infection. Intriguingly, Mn2+ conspicuously stimulated lysosomal activity, as evidenced by hyperactivation of mTORC1 and increased endo/lysosomal acidity. After HBV-triggered internalization, the NTCP receptor was sorted to late endosomal compartments by the ESCRT machinery in concert with the invading virion. The establishment of HBV infection was found to be independent of lysosomal fusion-driven late endosome maturation; Mn2+-induced lysosomal hyperfunction virtually impaired infection, suggesting that virions may gain cytosolic access directly from late endosomes. In contrast, suppression of lysosomal activity substantially enhanced HBV infection. Prolonged mTORC1 inactivation facilitated viral infection by depleting lysosomes and accelerating endocytic transport of virions. Notably, treatment with the natural steroidal alkaloid tomatidine recapitulated the effects of Mn2+ in stimulating lysosomal activity and exhibited potent anti-HBV activity in HepG2-NTCP cells and in proliferating human hepatocyte organoids. These findings provide new insights into the post-endocytosis events of HBV infection. The negative regulation of early HBV infection by endo/lysosomal activity makes it a promising target for antiviral therapies.

Autophagy plays a crucial role in the host response to Mycobacterium tuberculosis (Mtb) infection, yet the dynamics and regulation of autophagy induction on Mtb-containing vacuoles (MCVs) remain only partially understood. We employed time-lapse confocal microscopy to investigate the recruitment of LC3B (LC3), a key autophagy marker, to MCVs at the single cell level with our newly developed workflow for single cell and single MCV tracking and fluorescence quantification. We show that approximately 70% of MCVs exhibited LC3 recruitment but that was lost in about 40% of those MCVs. The LC3 recruitment to MCVs displayed a high variability in timing that was independent of the size of the MCV or the bacterial burden. Most notably, the LC3-positive MCVs did not acidify, indicating that LC3 recruitment does not necessarily lead to the formation of mature autophagolysosomes. Interferon-gamma pre-treatment did not affect LC3 recruitment frequency or autophagosome acidification but increased the susceptibility of the macrophage to Mtb-induced cell death. LC3 recruitment and lysotracker staining were mutually exclusive events, alternating on some MCVs multiple times thus demonstrating a reversible aspect of the autophagy response. The LC3 recruitment was associated with galectin-3 and oxysterol-binding protein 1 staining, indicating a correlation with membrane damage and repair mechanisms. ATG7 knock-down did not impact membrane repair, suggesting that autophagy is not directly involved in this process but is coregulated by the membrane damage of MCVs. In summary, our findings provide novel insights into the dynamic and variable nature of LC3 recruitment to the MCVs over time during Mtb infection. Our data does not support a role for autophagy in either cell-autonomous defense against Mtb or membrane repair of the MCV in human macrophages. In addition, the combined dynamics of LC3 recruitment and Lysoview staining emerged as promising markers for investigating the damage and repair processes of phagosomal membranes.

Neisseria gonorrhoeae exhibits alarming antibiotic resistance trends and poses a significant challenge in therapeutic management. This study aimed to explore the association of penA alleles with penicillin-binding protein (PBP) occupancy patterns and reduced outer membrane permeability, impacting susceptibility to last-line cephalosporins and potential β-lactam candidates. The whole genome sequence, the MICs and PBP IC50s were determined for 12 β-lactams and β-lactamase inhibitors in 8 clinical isolates with varying β-lactam sensitivity, 2 ATCC, and 3 WHO cephalosporin-resistant reference strains. The genetic analysis identified diverse determinants of β-lactam resistance including penA, ponA, porB, and mtrR alterations. Mosaic penA alleles were confirmed to be key determinants of cephalosporin resistance, with notable impacts on PBP2 IC50 affinities (in the presence of all PBPs). Substitutions in positions V316 and A501 exhibited significant effects on β-lactam PBP2 occupancy and MICs. PBP1 inhibition showed marginal effect on β-lactam sensitivity and PBP3 acted as a sink target. Ertapenem and piperacillin emerged as potential therapies against cephalosporin-resistant N. gonorrhoeae strains, along with combination therapies involving tazobactam and/or efflux inhibitors. The study determined the β-lactam PBP-binding affinities of last-line cephalosporins and alternative β-lactam candidates in strains carrying different penA alleles for the first time. These findings provide insights for developing new antimicrobial agents and enhancers against emerging resistant strains. Further research is warranted to optimize therapeutic interventions for cephalosporin-resistant N. gonorrhoeae infections.

Pathogenesis of Toxoplasma gondii in the intermediate host is based on the tachyzoite ability to divide rapidly to produce significant amount of daughter cells in a reduce time frame. The regulation of the cell-cycle specific expression program is therefore key to their proliferation. Transcriptional regulation has a crucial role in establishing this expression program and transcription factors regulates many aspects of tachyzoite cell cycle. We explored the role of two ApiAP2 transcription factors, TgAP2XII-9 and TgAP2III-2, during the cell cycle of the tachyzoite form. While TgAP2III-2 has only a minor impact on the tachyzoite proliferation, we show that TgAP2XII-9 regulates many aspects of the cell cycle including the proper assembly of the daughter cells inner membrane complex and temporal expression of many virulence genes. Creation of a double mutant strain for TgAP2XII-9 and TgAP2III-2 shows that TgAP2XII-9 had a prominent role during daughter cell assembly. Using transcriptomics and Cut&Tag, we demonstrate that TgAP2XII-9 mainly acts through the transcriptional control of at least 300 genes promoters. Interestingly, TgAP2XII-9 plays a crucial role repressing the expression of genes necessary for budding initiation and activating genes necessary for microneme de novo formation. We also explored the importance of the AP2 domain of TgAP2XII-9 demonstrating its critical role to exert its function. Therefore, we showed that TgAP2XII-9 is a crucial transcription factor which is key to daughter cell assembly post budding initiation.

Borrelia (or Borreliella) burgdorferi, the causative agent of Lyme disease, is a motile and invasive zoonotic pathogen adept at navigating between its arthropod vector and mammalian host. While motility and chemotaxis are well known to be essential for its enzootic cycle, the role of each methyl-accepting chemotaxis proteins (MCPs) in the infectious cycle of B. burgdorferi remains unclear. In this study, we show that mcp5, a gene encoding one of the most abundant MCPs in B. burgdorferi, is differentially expressed in response to environmental signals and at distinct stages of the pathogen's enzootic cycle. Notably, mcp5 expression is regulated by the Hk1-Rrp1 and Rrp2-RpoN-RpoS pathways, two key regulatory pathways that are critical for the spirochete's colonization of the tick vector and mammalian host, respectively. Infection experiments with an mcp5 mutant revealed that spirochetes lacking MCP5 were unable to establish infections in either C3H/HeN mice or Severe Combined Immunodeficiency (SCID) mice, which are deficient in adaptive immunity, underscoring MCP5's critical role in mammalian infection. However, the mcp5 mutant was able to establish infection and disseminate in NOD SCID Gamma (NSG) mice, which are deficient in both adaptive and most innate immune responses, suggesting that MCP5 plays an important role in evading host innate immunity. Moreover, NK cell depletion in C3H and SCID mice restored the infectivity of the mcp5 mutant, further highlighting MCP5's role in evading NK cell-associated immunity. Co-culture assays with NK cells and macrophages revealed that the mcp5 mutant enhanced interferon-gamma production by NK cells. In the tick vector, the mcp5 mutants survived feeding but failed to transmit to mice. These findings reveal that MCP5, regulated by both the Rrp1 and Rrp2 pathways, is critical for establishing infection in mammalian hosts by evading NK cell-mediated host innate immunity and is important for the transmission of spirochetes from ticks to mammalian hosts. This work provides a foundation for further elucidation of chemotactic signals sensed by MCP5 that facilitate B. burgdorferi in evading host defenses.