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Aspects of transcriptional regulation of plant cell wall-degrading enzyme (PCWDE) genes have been characterized in the filamentous fungus Neurospora crassa. However, the upstream signaling pathways that regulate PCWDE expression are not well understood. We have previously reported roles for heterotrimeric G-proteins and adenylyl cyclase in the degradation of cellulose to glucose in N. crassa. Here, we performed mRNA-seq to identify patterns of gene expression after transfer from glucose to cellulose medium in wild type, the Gα mutants Δgna-1 and Δgna-3, and the adenylyl cyclase mutant Δcr-1. In wild type, 3719 genes were regulated at least twofold during growth on cellulose vs glucose. Analysis of transcriptomics data for the strains after transfer from glucose to cellulose demonstrated that the Δcr-1 mutant had the most misregulated genes, with 2,232, followed by Δgna-3 with 1,182 and Δgna-1 with 648 genes. Metabolic genes were the most prevalent differentially expressed genes in the mutants. Expression of PCWDEs, including most of the cellulases, was downregulated in the three mutants, with Δcr-1 displaying the greatest deficiency. Furthermore, several transcription factors essential for cellulase expression were misregulated in the mutants. The primary factors clr-1 and clr-2 were downregulated in Δgna-3 and Δcr-1 strains, and clr-2 was reduced in Δgna-1 mutants. Overexpression of clr-2 restored cellulase activity and increased the expression of two major cellulase genes in all three mutants. Taken together, our results demonstrate that heterotrimeric G-proteins and cAMP signaling strongly impact transcriptional control of cellulase activity, culminating in the expression of the transcription factor clr-2 in N. crassa.IMPORTANCEFilamentous fungi are important organisms for degradation of plant biomass. Both nonpathogens and plant pathogens secrete plant cell wall degrading enzymes to release simple sugars from the plant cell wall to use as carbon sources for growth. Much is known about the transcription factors that control production of plant cell wall-degrading enzymes by fungi. However, mechanistic details for how different lignocellulosic compounds are sensed by these organisms and the resultant cellular responses that operate upstream of cellulase-regulating transcription factors are lacking. Our research helps bridge this gap by identifying the role of G-protein subunits and cAMP in the regulation of gene expression during growth on cellulose. Understanding the environmental sensing and signal transduction pathways that regulate cellulase gene expression will have applications to agricultural losses due to plant pathogens, carbon recycling in the environment, and production of biofuels.

Adeno-associated virus (AAV) vectors have taken center stage for gene therapy and have shown clinical efficacy in 15 human diseases to date. The Food and Drug Administration has approved seven AAV "drugs" for one-time treatment respectively for Leber's congenital amaurosis, spinal muscular atrophy, hemophilia B, Duchenne muscular dystrophy, hemophilia A, and aromatic L-amino acid decarboxylase deficiency. Despite these remarkable developments, it has become increasingly clear that the first generation of AAV vectors is less than optimal since in most, if not all, cases, exceedingly high doses are needed to achieve clinical efficacy, and as a consequence, in some patients, serious adverse events have been observed, and to date, at least 21 patients have died. Thus, there is a need to reassess the limitations of the first generation of AAV vectors as well as an urgent need to develop the next generation of AAV vectors that are safe and effective.

Salmonella enterica serovar (S.) Typhi, the etiological agent of typhoid fever, is strictly human adapted, which presents a significant challenge for studying its pathogenesis in animal models. A common strategy to overcome this limitation is to infect mice with S. Typhimurium as a surrogate pathogen. Since S. Typhimurium is a non-typhoidal serovar that does not encode the virulence-associated capsular polysaccharide (Vi antigen) of S. Typhi, we explored whether the mouse virulent typhoidal Salmonella serovar Paratyphi C, which expresses the Vi antigen, would be better suited as a surrogate pathogen to study typhoid fever pathogenesis in the mouse. In contrast to the nontyphoidal serovar Typhimurium, which produced lethal morbidity in C57BL/6 mice within a few days after infection, S. Paratyphi C demonstrated prolonged colonization of systemic organs for up to 28 days after infection. Analysis of virulence factors revealed that the Vi antigen was important at very early stages after infection (up to 2 days), whereas the type III secretion system encoded by Salmonella pathogenicity island 2 became critical at later stages. Vaccination with purified Vi antigen suppressed S. Paratyphi C dissemination. Implantation of a biotelemetry device revealed that S. Paratyphi C triggered fever after an incubation period of 3 days, which was reminiscent of the prolonged incubation period of typhoid fever. In conclusion, our findings suggest that the use of S. Paratyphi C as a surrogate pathogen provides a mouse model for studying typhoid fever pathogenesis and vaccine development.IMPORTANCEThe emergence of extensively drug-resistant Salmonella enterica serovar (S.) Typhi poses a serious threat to public health, but its host restriction to humans poses a challenge for studying pathogenesis and vaccine development in animal models. Here, we used S. Paratyphi C, a mouse virulent typhoidal serovar that expresses the virulence-associated Vi capsular polysaccharide, as a surrogate pathogen for studying typhoid fever in a mouse model. Our model recapitulates key features of typhoid fever, including clinical symptoms such as a prolonged incubation period, fever, and splenomegaly. Notably, disseminated infection with S. Paratyphi C developed after inoculation by the natural oral route. We demonstrate the utility of this model for studying pathogenesis and vaccination. We conclude that our new mouse model for typhoid fever offers a promising platform for evaluating novel therapeutics and vaccine candidates to address the problem of drug resistance in S. Typhi and reduce the global burden of typhoid fever.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that is a potent inducer of cell-mediated immunity, which has led to the development of attenuated, Listeria-based cancer vaccines. L. monocytogenes strains, such as live-attenuated double-deleted Listeria (LADD), lacking two key virulence factors, ΔactA and ΔinlB, have been used safely in clinical trials and showed promising anti-tumor activity. Despite early clinical success, improving potency and safety by preventing extracellular bacterial growth is paramount for the development of further clinical applications. We describe a quadruple attenuated intracellular Listeria (QUAIL) strain that, in addition to ΔactAΔinlB, lacks ribC and ribF, which encode enzymes required for generating the essential flavin cofactors flavin mononucleotide (FMN) and flavin adenine nucleotide (FAD). QUAIL imported FMN and FAD during intracellular growth but was unable to grow extracellularly in blood or on vascular catheters in mice, which reduced its lethality. Despite its lack of extracellular growth, QUAIL maintained its immunoprotective properties, which were comparable to LADD. Furthermore, we showed that QUAIL can be engineered to synthesize riboflavin, leading to expansion and activation of mucosal-associated invariant T cells. Together, our data support the use of QUAIL as a promising therapeutic platform with an improved safety profile that is amenable to further modifications to expand its immune-activating potential.IMPORTANCEListeria-based live-attenuated cancer vaccines represent a promising therapy in many different pre-clinical tumor models and in clinical trials. Enhancing its anti-cancer immunity and increasing its safety profile will advance the clinical applications of Listeria vaccines. By manipulating Listeria monocytogenes flavin metabolism, we engineered a quadruple attenuated intracellular Listeria (QUAIL) vaccine candidate strain that has limited toxicity associated with extracellular growth in major extracellular niches in vivo, including blood and implanted catheter ports. Furthermore, we showed that QUAIL can be effectively programmed to engage innate-like T cells known as mucosal-associated invariant T cells, which could be harnessed for future cancer immunotherapies. The results presented here lay the foundation for further analysis of QUAIL as a safer, yet immunopotent L. monocytogenes vaccine or therapeutic vector.

Swine enteric coronaviruses (SeCoVs), including transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and porcine deltacoronavirus (PDCoV), have been reported to use aminopeptidase N (APN) as a cellular receptor. However, APN alone cannot effectively explain the infection of both APN-positive and APN-negative enterocytes by PEDV and TGEV, nor the wide host range of PDCoV, suggesting the involvement of other host factors. In this study, we demonstrate that TGEV infection in piglets upregulates claudin-1 expression not only in infected cells but also in uninfected cells. Claudin-1 levels correlated strongly with TGEV N protein levels in the jejunum of infected piglets. Functional studies revealed that claudin-1 overexpression enhanced cellular susceptibility to TGEV, PEDV, and PDCoV, whereas its knockout significantly attenuated infection. Mechanistically, claudin-1 specifically interacts with the S1 or receptor-binding domain (RBD) of SeCoVs and promotes viral internalization. Furthermore, induction of claudin-1 in piglets promotes PDCoV infection in the intestine. Notably, claudin-1 also binds to the S1 protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Collectively, our results identify claudin-1 as a novel internalization factor for porcine enteric coronaviruses, playing a critical role in facilitating infection within the digestive tract, and highlight its potential as a target for future clinical interventions.

Importance: We observed a downregulation in the expression of the majority of tight junction proteins in intestinal tissues infected with transmissible gastroenteritis virus (TGEV). However, unexpectedly, claudin-1 exhibited a significant upregulation in intestinal epithelial cells. This intriguing finding prompted us to delve deeper into the potential role of claudin-1 in facilitating virus invasion of epithelial cells. Utilizing overexpression and knockout cell lines, we demonstrate that claudin-1 is an internalization factor for swine enteric coronaviruses (SeCoVs), including TGEV, porcine epidemic diarrhea virus (PEDV), and porcine deltacoronavirus (PDCoV). Notably, claudin-1 interacts with the S1 protein of TGEV, PEDV, PDCoV, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spanning across alpha, beta, and delta coronaviruses. Our findings provide deeper insights into the infection mechanisms and pathogenesis of SeCoVs and SARS-CoV-2.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging bat-origin alphacoronavirus causing severe disease in neonatal piglets, with significant economic losses to the swine industry. The virus exhibits a broad species tropism, infecting cells derived from pigs, humans, and mice, highlighting its potential for cross-species transmission. Due to drawbacks associated with the use of young piglets, there is a need for an appropriate small animal model to study SADS-CoV biology. Here we established a mouse infection model based on a murinized mutant of the virus, mSADS-CoV, in which the ectodomain of the viral spike protein was replaced by that of the murine coronavirus mouse hepatitis virus. This chimeric virus, generated through targeted RNA recombination, replicated efficiently in murine cell cultures and exhibited an age-dependent infection in neonatal mice that was lethal in 2-day-old BALB/c mice, affecting various organs, notably the intestine. We validated our infection model by successfully verifying the efficacy of the RNA-dependent RNA polymerase inhibitor remdesivir. The model will serve as a valuable tool for studying SADS-CoV pathogenesis and for elucidating the roles of host factors in viral replication as well as for preclinical evaluation of antiviral compounds targeting the viral replication machinery.IMPORTANCESwine acute diarrhea syndrome coronavirus (SADS-CoV) poses a threat to the swine industry and public health because of its broad species tropism and potential for cross-species transmission. The emergence of other bat-derived coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and Middle East respiratory syndrome coronavirus, underscores the need for robust models to study these pathogens. The successful rescue of mSADS-CoV and the development of a mouse infection model represent significant advancements in SADS-CoV research. This model not only enables the evaluation of antiviral therapeutics such as remdesivir but also provides a powerful platform for investigating viral replication mechanisms and host-pathogen interactions, offering critical insights for pandemic preparedness.