Severe fever with thrombocytopenia syndrome (SFTS) is a rapidly progressing infectious disease caused by a novel bunyavirus characterized by high fever, thrombocytopenia, and multiple organ damage. While lipids play an important role in viral infections, the specific alterations in lipid metabolism during SFTSV infection remain unclear. This study aimed to elucidate the global lipid metabolic profiles of SFTS patients with mild, severe, and fatal outcomes. A total of 60 SFTS patients, consisting of 30 mild, 15 severe and 15 fatal patients, and 30 healthy controls, were enrolled for the investigation of global lipidomics in serum using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our findings revealed global alterations in the lipid signature induced by SFTSV infection and further confirmed that the glycerophospholipid metabolism pathway was profoundly affected during the progression of mild, severe, and fatal outcomes in SFTS patients. Importantly, LysoPC (20:0) and LysoPC (P-16:0) are strongly correlated with the clinical parameters of SFTSV infection. Furthermore, we demonstrated the substantial prognostic value of LysoPC (20:0) and LysoPC (P-16:0) by receiver operating characteristic (ROC) curve analysis, providing evidence for their remarkable value as prognostic biomarkers for predicting SFTS clinical outcomes. In particular, LysoPC (20:0) and LysoPC (P-16:0), along with APTT, yielded superior prognostic performance for fatal SFTS [area under the curve (AUC) = 98.4%], outperforming routine clinical parameters. Collectively, our findings revealed the lipidomic landscape after SFTSV infection, which offers new insights into the mechanisms of SFTS disease progression and suggests that targeting lipid metabolism may serve as a potential therapeutic strategy.
Importance: This study systematically investigated the lipid landscape profile of SFTS-infected patients with different clinical outcomes. Our results revealed a global alteration in the lipid signature, particularly the glycerophospholipid metabolic pathway, induced by SFTSV infection. Notably, LysoPC (20:0) and LysoPC (P-16:0) presented remarkable prognostic value as novel biomarkers for SFTSV infection and may contribute to the prognosis of SFTS progression and appropriate interventions.
{"title":"A global lipid map of severe fever with thrombocytopenia syndrome virus infection reveals glycerophospholipids as novel prognosis biomarkers.","authors":"Panpan Tian, Liwei Zhao, Guiting Zhang, Shixing Chen, Wanying Zhang, Mingrong Ou, Yidan Sun, Yuxin Chen","doi":"10.1128/mbio.02628-24","DOIUrl":"https://doi.org/10.1128/mbio.02628-24","url":null,"abstract":"<p><p>Severe fever with thrombocytopenia syndrome (SFTS) is a rapidly progressing infectious disease caused by a novel bunyavirus characterized by high fever, thrombocytopenia, and multiple organ damage. While lipids play an important role in viral infections, the specific alterations in lipid metabolism during SFTSV infection remain unclear. This study aimed to elucidate the global lipid metabolic profiles of SFTS patients with mild, severe, and fatal outcomes. A total of 60 SFTS patients, consisting of 30 mild, 15 severe and 15 fatal patients, and 30 healthy controls, were enrolled for the investigation of global lipidomics in serum using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our findings revealed global alterations in the lipid signature induced by SFTSV infection and further confirmed that the glycerophospholipid metabolism pathway was profoundly affected during the progression of mild, severe, and fatal outcomes in SFTS patients. Importantly, LysoPC (20:0) and LysoPC (P-16:0) are strongly correlated with the clinical parameters of SFTSV infection. Furthermore, we demonstrated the substantial prognostic value of LysoPC (20:0) and LysoPC (P-16:0) by receiver operating characteristic (ROC) curve analysis, providing evidence for their remarkable value as prognostic biomarkers for predicting SFTS clinical outcomes. In particular, LysoPC (20:0) and LysoPC (P-16:0), along with APTT, yielded superior prognostic performance for fatal SFTS [area under the curve (AUC) = 98.4%], outperforming routine clinical parameters. Collectively, our findings revealed the lipidomic landscape after SFTSV infection, which offers new insights into the mechanisms of SFTS disease progression and suggests that targeting lipid metabolism may serve as a potential therapeutic strategy.</p><p><strong>Importance: </strong>This study systematically investigated the lipid landscape profile of SFTS-infected patients with different clinical outcomes. Our results revealed a global alteration in the lipid signature, particularly the glycerophospholipid metabolic pathway, induced by SFTSV infection. Notably, LysoPC (20:0) and LysoPC (P-16:0) presented remarkable prognostic value as novel biomarkers for SFTSV infection and may contribute to the prognosis of SFTS progression and appropriate interventions.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0262824"},"PeriodicalIF":5.1,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142623708","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The assembly and function of side chain modifications of glycosylphosphatidylinositol (GPI) units (anchors or free forms) are poorly defined. In a recent study, two enzymes, PIGJ and PIGE, of the protozoan parasite Toxoplasma gondii were identified and shown to be involved in the assembly of such GPI side chains (J. A. Alvarez, E. Gas-Pascual, S. Malhi, J. C. Sánchez-Arcila, et al., mBio 15:e00527-24, 2024, https://doi.org/10.1128/mbio.00527-24). PIGJ adds N-acetylgalactosamine to the GPI core structure, while PIGE subsequently adds a terminal glucose. Deletion of PIGJ resulted in the loss of the side chain and, strikingly, increased mortality in infected mice, in contrast to PIGE knockouts. Absence of the side chain led to increased binding of the scavenger receptor CD36 to mutant parasites. In galectin-3 knockout mice, the virulent phenotype of side-chain-deficient parasites was largely lost. While the exact mechanisms remain to be elucidated by more experiments, these findings provide the first evidence for the importance of GPI side chains in parasite-host interactions in vivo.
糖基磷脂酰肌醇(GPI)单位(锚或游离形式)侧链修饰的组装和功能尚不明确。在最近的一项研究中,确定了原生动物寄生虫弓形虫的两种酶 PIGJ 和 PIGE,并证明它们参与了此类 GPI 侧链的组装(J. A. Alvarez, E. Gas-Pascual, S. Malhi, J. C. Sánchez-Arcila, et al., mBio 15:e00527-24, 2024, https://doi.org/10.1128/mbio.00527-24)。PIGJ 向 GPI 核心结构添加 N-乙酰半乳糖胺,而 PIGE 随后添加末端葡萄糖。与 PIGE 基因敲除相反,PIGJ 基因缺失会导致侧链缺失,并显著增加受感染小鼠的死亡率。侧链的缺失导致清道夫受体 CD36 与突变寄生虫的结合增加。在 galectin-3 基因敲除小鼠中,侧链缺陷寄生虫的毒性表型基本消失。虽然确切的机制还有待更多的实验来阐明,但这些发现首次证明了 GPI 侧链在寄生虫-宿主体内相互作用中的重要性。
{"title":"When more sugar is better-a GPI side chain modification results in a less virulent phenotype during a protozoan infection.","authors":"Frank Seeber","doi":"10.1128/mbio.02740-24","DOIUrl":"https://doi.org/10.1128/mbio.02740-24","url":null,"abstract":"<p><p>The assembly and function of side chain modifications of glycosylphosphatidylinositol (GPI) units (anchors or free forms) are poorly defined. In a recent study, two enzymes, PIGJ and PIGE, of the protozoan parasite <i>Toxoplasma gondii</i> were identified and shown to be involved in the assembly of such GPI side chains (J. A. Alvarez, E. Gas-Pascual, S. Malhi, J. C. Sánchez-Arcila, et al., mBio 15:e00527-24, 2024, https://doi.org/10.1128/mbio.00527-24). PIGJ adds N-acetylgalactosamine to the GPI core structure, while PIGE subsequently adds a terminal glucose. Deletion of PIGJ resulted in the loss of the side chain and, strikingly, increased mortality in infected mice, in contrast to PIGE knockouts. Absence of the side chain led to increased binding of the scavenger receptor CD36 to mutant parasites. In galectin-3 knockout mice, the virulent phenotype of side-chain-deficient parasites was largely lost. While the exact mechanisms remain to be elucidated by more experiments, these findings provide the first evidence for the importance of GPI side chains in parasite-host interactions <i>in vivo</i>.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0274024"},"PeriodicalIF":5.1,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142622413","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-13Epub Date: 2024-10-15DOI: 10.1128/mbio.02374-24
Daniel Sauter, Frank Kirchhoff
Guanylate-binding proteins (GBPs) are interferon-inducible cellular factors known to inhibit a wide variety of pathogens. Humans encode seven GBPs that have functionally diversified to provide broad protection against a variety of bacteria, protozoa, and viruses. Here, we discuss recent data on the mechanisms underlying the broad antiviral activity of GBP5 (H. Veler, C. M. Lun, A. A. Waheed, and E. O. Freed, mBio e02086-24, 2024, https://doi.org/10.1128/mbio.02086-24) and place them in the context of previous studies on the ability of this antiviral factor to impair the function of numerous viral envelope (Env) glycoproteins. We focus on the effects of GBP5 on the glycosylation, proteolytic processing, and anterograde transport of Env and discuss mechanistic interdependencies of these maturation steps. Understanding the induction and action of broadly acting immune factors, such as GBP5, may help develop effective immune-based strategies against numerous pathogens.
鸟苷酸结合蛋白(GBPs)是干扰素诱导的细胞因子,已知可抑制多种病原体。人类编码了七种 GBPs,它们在功能上已经多样化,能够提供广泛的保护,抵御各种细菌、原生动物和病毒。在此,我们讨论了有关 GBP5 广泛抗病毒活性机制的最新数据(H. Veler、C. M. Lun、A. A. Waheed 和 E. O. Freed,mBio e02086-24,2024,https://doi.org/10.1128/mbio.02086-24),并将这些数据与之前有关这种抗病毒因子能够损害多种病毒包膜(Env)糖蛋白功能的研究相结合。我们重点研究了 GBP5 对 Env 的糖基化、蛋白水解加工和逆向运输的影响,并讨论了这些成熟步骤的机理相互依存关系。了解 GBP5 等广泛作用的免疫因子的诱导和作用可能有助于开发有效的免疫策略来对抗多种病原体。
{"title":"Antiviral mechanisms of guanylate-binding protein 5: versatile inhibition of multiple viral glycoproteins.","authors":"Daniel Sauter, Frank Kirchhoff","doi":"10.1128/mbio.02374-24","DOIUrl":"10.1128/mbio.02374-24","url":null,"abstract":"<p><p>Guanylate-binding proteins (GBPs) are interferon-inducible cellular factors known to inhibit a wide variety of pathogens. Humans encode seven GBPs that have functionally diversified to provide broad protection against a variety of bacteria, protozoa, and viruses. Here, we discuss recent data on the mechanisms underlying the broad antiviral activity of GBP5 (H. Veler, C. M. Lun, A. A. Waheed, and E. O. Freed, mBio e02086-24, 2024, https://doi.org/10.1128/mbio.02086-24) and place them in the context of previous studies on the ability of this antiviral factor to impair the function of numerous viral envelope (Env) glycoproteins. We focus on the effects of GBP5 on the glycosylation, proteolytic processing, and anterograde transport of Env and discuss mechanistic interdependencies of these maturation steps. Understanding the induction and action of broadly acting immune factors, such as GBP5, may help develop effective immune-based strategies against numerous pathogens.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0237424"},"PeriodicalIF":5.1,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11559056/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142469436","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-13Epub Date: 2024-10-17DOI: 10.1128/mbio.02578-24
Jesus M Eraso, Randall J Olsen, S Wesley Long, Ryan Gadd, Sarrah Boukthir, Ahmad Faili, Samer Kayal, James M Musser
Streptococcus dysgalactiae subspecies equisimilis (SDSE) is a Gram-positive bacterial pathogen that infects humans and is closely related to group A streptococcus (GAS). Compared with GAS, far less is known about SDSE pathobiology. Increased rates of invasive SDSE infections have recently been reported in many countries. One SDSE emm type (stG62647) is known to cause severe diseases, including necrotizing soft-tissue infections, endocarditis, and osteoarticular infections. To increase our understanding of the molecular pathogenesis of stG62647 SDSE isolates causing human infections, we sequenced to closure the genomes of 120 stG62647 SDSE isolates. The genomes varied in size from 2.1 to 2.24 Mb pairs. The great majority of stG62647 isolates had IS1548 integrated into the silB gene, thereby inactivating it. Regions of difference, such as mobile genetic elements, were the largest source of genomic diversity. All 120 stG62647 isolates were assayed for virulence using a well-established mouse model of necrotizing myositis. An unexpectedly wide range of virulence was identified (20% to 95%), as assessed by near-mortality data. To explore the molecular mechanisms underlying virulence differences, we analyzed RNAseq transcriptome profiles for 38 stG62647 isolates (comprising the 19 least and most virulent) grown in vitro. Genetic polymorphisms were identified from whole-genome sequence data. Collectively, the results suggest that these SDSE isolates use multiple genetic pathways to alter virulence phenotype. The data also suggest that human genetics and underlying medical conditions contribute to disease severity. Our study integrates genomic, mouse virulence, and RNAseq data to advance our understanding of SDSE pathobiology and its molecular pathogenesis.
Importance: This study integrated genomic sequencing, mouse virulence assays, and bacterial transcriptomic analysis to advance our understanding of the molecular mechanisms contributing to Streptococcus dysgalactiae subsp. equisimilis emm type stG62647 pathogenesis. We tested a large cohort of genetically closely related stG62647 isolates for virulence using an established mouse model of necrotizing myositis and discovered a broad spectrum of virulence phenotypes, with near-mortality rates ranging from 20% to 95%. This variation was unexpected, given their close genetic proximity. Transcriptome analysis of stG62647 isolates responsible for the lowest and highest near-mortality rates suggested that these isolates used multiple molecular pathways to alter their virulence. In addition, some genes encoding transcriptional regulators and putative virulence factors likely contribute to SDSE emm type stG62647 pathogenesis. These data underscore the complexity of pathogen-host interactions in an emerging SDSE clonal group.
{"title":"Integrative genomic, virulence, and transcriptomic analysis of emergent <i>Streptococcus dysgalactiae</i> subspecies <i>equisimilis</i> (SDSE) <i>emm</i> type <i>stG62647</i> isolates causing human infections.","authors":"Jesus M Eraso, Randall J Olsen, S Wesley Long, Ryan Gadd, Sarrah Boukthir, Ahmad Faili, Samer Kayal, James M Musser","doi":"10.1128/mbio.02578-24","DOIUrl":"10.1128/mbio.02578-24","url":null,"abstract":"<p><p><i>Streptococcus dysgalactiae</i> subspecies <i>equisimilis</i> (SDSE) is a Gram-positive bacterial pathogen that infects humans and is closely related to group A streptococcus (GAS). Compared with GAS, far less is known about SDSE pathobiology. Increased rates of invasive SDSE infections have recently been reported in many countries. One SDSE <i>emm</i> type (<i>stG62647</i>) is known to cause severe diseases, including necrotizing soft-tissue infections, endocarditis, and osteoarticular infections. To increase our understanding of the molecular pathogenesis of <i>stG62647</i> SDSE isolates causing human infections, we sequenced to closure the genomes of 120 <i>stG62647</i> SDSE isolates. The genomes varied in size from 2.1 to 2.24 Mb pairs. The great majority of <i>stG62647</i> isolates had <i>IS1548</i> integrated into the <i>silB</i> gene, thereby inactivating it. Regions of difference, such as mobile genetic elements, were the largest source of genomic diversity. All 120 <i>stG62647</i> isolates were assayed for virulence using a well-established mouse model of necrotizing myositis. An unexpectedly wide range of virulence was identified (20% to 95%), as assessed by near-mortality data. To explore the molecular mechanisms underlying virulence differences, we analyzed RNAseq transcriptome profiles for 38 <i>stG62647</i> isolates (comprising the 19 least and most virulent) grown <i>in vitro</i>. Genetic polymorphisms were identified from whole-genome sequence data. Collectively, the results suggest that these SDSE isolates use multiple genetic pathways to alter virulence phenotype. The data also suggest that human genetics and underlying medical conditions contribute to disease severity. Our study integrates genomic, mouse virulence, and RNAseq data to advance our understanding of SDSE pathobiology and its molecular pathogenesis.</p><p><strong>Importance: </strong>This study integrated genomic sequencing, mouse virulence assays, and bacterial transcriptomic analysis to advance our understanding of the molecular mechanisms contributing to <i>Streptococcus dysgalactiae</i> subsp. <i>equisimilis emm</i> type <i>stG62647</i> pathogenesis. We tested a large cohort of genetically closely related <i>stG62647</i> isolates for virulence using an established mouse model of necrotizing myositis and discovered a broad spectrum of virulence phenotypes, with near-mortality rates ranging from 20% to 95%. This variation was unexpected, given their close genetic proximity. Transcriptome analysis of <i>stG62647</i> isolates responsible for the lowest and highest near-mortality rates suggested that these isolates used multiple molecular pathways to alter their virulence. In addition, some genes encoding transcriptional regulators and putative virulence factors likely contribute to SDSE <i>emm</i> type <i>stG62647</i> pathogenesis. These data underscore the complexity of pathogen-host interactions in an emerging SDSE clonal group.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0257824"},"PeriodicalIF":5.1,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11559094/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142469440","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-13Epub Date: 2024-10-03DOI: 10.1128/mbio.01206-24
Alessandra Lo Sciuto, Francesca D'Angelo, Maria Concetta Spinnato, Pierre Simon Garcia, Shirley Genah, Cervoni Matteo, Emmanuel Séchet, Ehud Banin, Frédéric Barras, Francesco Imperi
Iron-sulfur [Fe-S] clusters are essential protein cofactors allowing bacteria to perceive environmental redox modification and to adapt to iron limitation. Escherichia coli, which served as a bacterial model, contains two [Fe-S] cluster biogenesis systems, ISC and SUF, which ensure [Fe-S] cluster synthesis under balanced and stress conditions, respectively. However, our recent phylogenomic analyses revealed that most bacteria possess only one [Fe-S] cluster biogenesis system, most often SUF. The opportunist human pathogen Pseudomonas aeruginosa is atypical as it harbors only ISC. Here, we confirmed the essentiality of ISC in P. aeruginosa under both normal and stress conditions. Moreover, P. aeruginosa ISC restored viability, under balanced growth conditions, to an E. coli strain lacking both ISC and SUF. Reciprocally, the E. coli SUF system sustained growth and [Fe-S] cluster-dependent enzyme activities of ISC-deficient P. aeruginosa. Surprisingly, an ISC-deficient P. aeruginosa strain expressing E. coli SUF showed defects in resistance to H2O2 stress and paraquat, a superoxide generator. Similarly, the P. aeruginosa ISC system did not confer stress resistance to a SUF-deficient E. coli mutant. A survey of 120 Pseudomonadales genomes confirmed that all but five species have selected ISC over SUF. While highlighting the great versatility of bacterial [Fe-S] cluster biogenesis systems, this study emphasizes that their contribution to cellular homeostasis must be assessed in the context of each species and its own repertoire of stress adaptation functions. As a matter of fact, despite having only one ISC system, P. aeruginosa shows higher fitness in the face of ROS and iron limitation than E. coli.
Importance: ISC and SUF molecular systems build and transfer Fe-S cluster to cellular apo protein clients. The model Escherichia coli has both ISC and SUF and study of the interplay between the two systems established that the ISC system is the house-keeping one and SUF the stress-responding one. Unexpectedly, our recent phylogenomic analysis revealed that in contrast to E. coli (and related enterobacteria such as Salmonella), most bacteria have only one system, and, in most cases, it is SUF. Pseudomonas aeruginosa fits the general rule of having only one system but stands against the rule by having ISC. This study aims at engineering P. aeruginosa harboring E. coli systems and vice versa. Comparison of the recombinants allowed to assess the functional versatility of each system while appreciating their contribution to cellular homeostasis in different species context.
{"title":"A molecular comparison of [Fe-S] cluster-based homeostasis in <i>Escherichia coli</i> and <i>Pseudomonas aeruginosa</i>.","authors":"Alessandra Lo Sciuto, Francesca D'Angelo, Maria Concetta Spinnato, Pierre Simon Garcia, Shirley Genah, Cervoni Matteo, Emmanuel Séchet, Ehud Banin, Frédéric Barras, Francesco Imperi","doi":"10.1128/mbio.01206-24","DOIUrl":"10.1128/mbio.01206-24","url":null,"abstract":"<p><p>Iron-sulfur [Fe-S] clusters are essential protein cofactors allowing bacteria to perceive environmental redox modification and to adapt to iron limitation. <i>Escherichia coli</i>, which served as a bacterial model, contains two [Fe-S] cluster biogenesis systems, ISC and SUF, which ensure [Fe-S] cluster synthesis under balanced and stress conditions, respectively. However, our recent phylogenomic analyses revealed that most bacteria possess only one [Fe-S] cluster biogenesis system, most often SUF. The opportunist human pathogen <i>Pseudomonas aeruginosa</i> is atypical as it harbors only ISC. Here, we confirmed the essentiality of ISC in <i>P. aeruginosa</i> under both normal and stress conditions. Moreover, <i>P. aeruginosa</i> ISC restored viability, under balanced growth conditions, to an <i>E. coli</i> strain lacking both ISC and SUF. Reciprocally, the <i>E. coli</i> SUF system sustained growth and [Fe-S] cluster-dependent enzyme activities of ISC-deficient <i>P. aeruginosa</i>. Surprisingly, an ISC-deficient <i>P. aeruginosa</i> strain expressing <i>E. coli</i> SUF showed defects in resistance to H<sub>2</sub>O<sub>2</sub> stress and paraquat, a superoxide generator. Similarly, the <i>P. aeruginosa</i> ISC system did not confer stress resistance to a SUF-deficient <i>E. coli</i> mutant. A survey of 120 Pseudomonadales genomes confirmed that all but five species have selected ISC over SUF. While highlighting the great versatility of bacterial [Fe-S] cluster biogenesis systems, this study emphasizes that their contribution to cellular homeostasis must be assessed in the context of each species and its own repertoire of stress adaptation functions. As a matter of fact, despite having only one ISC system, <i>P. aeruginosa</i> shows higher fitness in the face of ROS and iron limitation than <i>E. coli</i>.</p><p><strong>Importance: </strong>ISC and SUF molecular systems build and transfer Fe-S cluster to cellular apo protein clients. The model <i>Escherichia coli</i> has both ISC and SUF and study of the interplay between the two systems established that the ISC system is the house-keeping one and SUF the stress-responding one. Unexpectedly, our recent phylogenomic analysis revealed that in contrast to <i>E. coli</i> (and related enterobacteria such as Salmonella), most bacteria have only one system, and, in most cases, it is SUF. <i>Pseudomonas aeruginosa</i> fits the general rule of having only one system but stands against the rule by having ISC. This study aims at engineering <i>P. aeruginosa</i> harboring <i>E. coli</i> systems and vice versa. Comparison of the recombinants allowed to assess the functional versatility of each system while appreciating their contribution to cellular homeostasis in different species context.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0120624"},"PeriodicalIF":5.1,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11559095/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142365782","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
During a coronavirus infection, the spike protein undergoes sequential structural transitions triggered by its receptor and the host protease at the interface between the virus and cell membranes, thereby mediating membrane fusion. After receptor binding, the heptad repeat motif (HR1/HR2) within the viral spike protein bridges the viral and cellular membranes; however, the intermediate conformation adopted by the spike protein when drawing the viral and cellular membranes into close proximity remains unclear due to its transient and unstable nature. Here, we experimentally induced conformational changes in the spike protein of a murine coronavirus by incubating the virus with its receptor, followed by exposure to trypsin. We then treated the virus/receptor complex with proteinase K to probe the tightly packed core structure of the spike protein. The conformations of the spike protein were predicted from the sizes of the protease digestion products detected by western blot analysis. Upon receptor binding, two bands (each showing different reactivity with a fusion-inhibiting HR2-peptide) were detected; we propose that these bands correspond to the packed and unpacked HR1/HR2 motifs. After trypsin-mediated triggering, measurement of temperature and time dependency revealed that packing of the remaining unpacked HR1/HR2 motifs and assembly of three HR1 motifs in a trimer occur almost simultaneously. Thus, the trimeric spike protein adopts an asymmetric-unassembled conformation after receptor binding, followed by direct assembly into the post-fusion form triggered by the host protease. This biochemical study provides mechanistic insight into the previously unknown intermediate structure of the viral fusion protein.IMPORTANCEDuring infection by an enveloped virus, receptor binding triggers fusion between the cellular membrane and the virus envelope, enabling delivery of the viral genome to the cytoplasm. The viral spike protein mediates membrane fusion; however the molecular mechanism underlying this process is unclear. This is because using structural biology methods to track the transient conformational changes induced in the unstable spike trimer is challenging. Here, we harnessed the ability of protease enzymes to recognize subtle differences on protein surfaces, allowing us to detect structural differences in the spike protein before and after conformational changes. Differences in the size of the degradation products were analyzed by western blot analysis. The proposed model explaining the conformational changes presented herein is a plausible candidate that provides valuable insight into unanswered questions in the field of virology.
在冠状病毒感染过程中,尖峰蛋白在病毒和细胞膜界面上经历由其受体和宿主蛋白酶引发的连续结构转换,从而介导膜融合。受体结合后,病毒尖峰蛋白内的七重重复基序(HR1/HR2)将病毒膜和细胞膜连接起来;然而,由于尖峰蛋白的瞬时性和不稳定性,它将病毒膜和细胞膜拉近时所采用的中间构象仍不清楚。在这里,我们通过实验诱导了小鼠冠状病毒尖峰蛋白的构象变化,方法是将病毒与其受体孵育,然后暴露于胰蛋白酶。然后,我们用蛋白酶 K 处理病毒/受体复合物,以探究尖峰蛋白的紧密核心结构。尖峰蛋白的构象是通过蛋白酶消化产物的大小来预测的。受体结合后,检测到两条带(每条带与融合抑制 HR2 肽的反应性不同);我们认为这两条带对应于包装和未包装的 HR1/HR2 基序。在胰蛋白酶介导的触发后,温度和时间依赖性的测量显示,剩余未包装的 HR1/HR2 基序的包装和三个 HR1 基序在三聚体中的组装几乎同时发生。因此,三聚体尖峰蛋白在与受体结合后采用不对称的未组装构象,然后在宿主蛋白酶的触发下直接组装成融合后形式。这项生化研究从机理上揭示了病毒融合蛋白之前未知的中间结构。重要意义在包膜病毒感染期间,受体结合会触发细胞膜与病毒包膜之间的融合,从而将病毒基因组传递到细胞质中。病毒尖峰蛋白介导膜融合,但这一过程的分子机制尚不清楚。这是因为使用结构生物学方法来跟踪不稳定的尖峰三聚体中诱导的瞬时构象变化具有挑战性。在这里,我们利用蛋白酶识别蛋白质表面细微差别的能力,检测了构象变化前后尖峰蛋白的结构差异。降解产物的大小差异通过 Western 印迹分析进行了分析。本文提出的解释构象变化的模型是一个可信的候选模型,它为病毒学领域的未决问题提供了宝贵的见解。
{"title":"Biochemical analysis of packing and assembling heptad repeat motifs in the coronavirus spike protein trimer.","authors":"Jun Kobayashi, Kazuhiko Kanou, Hiyori Okura, Tahmina Mst Akter, Shuetsu Fukushi, Shutoku Matsuyama","doi":"10.1128/mbio.01203-24","DOIUrl":"10.1128/mbio.01203-24","url":null,"abstract":"<p><p>During a coronavirus infection, the spike protein undergoes sequential structural transitions triggered by its receptor and the host protease at the interface between the virus and cell membranes, thereby mediating membrane fusion. After receptor binding, the heptad repeat motif (HR1/HR2) within the viral spike protein bridges the viral and cellular membranes; however, the intermediate conformation adopted by the spike protein when drawing the viral and cellular membranes into close proximity remains unclear due to its transient and unstable nature. Here, we experimentally induced conformational changes in the spike protein of a murine coronavirus by incubating the virus with its receptor, followed by exposure to trypsin. We then treated the virus/receptor complex with proteinase K to probe the tightly packed core structure of the spike protein. The conformations of the spike protein were predicted from the sizes of the protease digestion products detected by western blot analysis. Upon receptor binding, two bands (each showing different reactivity with a fusion-inhibiting HR2-peptide) were detected; we propose that these bands correspond to the packed and unpacked HR1/HR2 motifs. After trypsin-mediated triggering, measurement of temperature and time dependency revealed that packing of the remaining unpacked HR1/HR2 motifs and assembly of three HR1 motifs in a trimer occur almost simultaneously. Thus, the trimeric spike protein adopts an asymmetric-unassembled conformation after receptor binding, followed by direct assembly into the post-fusion form triggered by the host protease. This biochemical study provides mechanistic insight into the previously unknown intermediate structure of the viral fusion protein.IMPORTANCEDuring infection by an enveloped virus, receptor binding triggers fusion between the cellular membrane and the virus envelope, enabling delivery of the viral genome to the cytoplasm. The viral spike protein mediates membrane fusion; however the molecular mechanism underlying this process is unclear. This is because using structural biology methods to track the transient conformational changes induced in the unstable spike trimer is challenging. Here, we harnessed the ability of protease enzymes to recognize subtle differences on protein surfaces, allowing us to detect structural differences in the spike protein before and after conformational changes. Differences in the size of the degradation products were analyzed by western blot analysis. The proposed model explaining the conformational changes presented herein is a plausible candidate that provides valuable insight into unanswered questions in the field of virology.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0120324"},"PeriodicalIF":5.1,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11559096/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142503521","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Christopher B Bullock, Leran Wang, Brian C Ware, Ngan Wagoner, Ray A Ohara, Tian-Tian Liu, Pritesh Desai, Bjoern Peters, Kenneth M Murphy, Scott A Handley, Thomas E Morrison, Michael S Diamond
Ross River virus (RRV) and other alphaviruses cause chronic musculoskeletal syndromes that are associated with viral persistence, which suggests deficits in immune clearance mechanisms, including CD8+ T-cell responses. Here, we used a recombinant RRV-gp33 that expresses the immunodominant CD8+ T-cell epitope of lymphocytic choriomeningitis virus (LCMV) to directly compare responses with a virus, LCMV, that strongly induces antiviral CD8+ T cells. After footpad injection, we detected fewer gp33-specific CD8+ T cells in the draining lymph node (DLN) after RRV-gp33 than LCMV infection, despite similar viral RNA levels in the foot. However, less RRV RNA was detected in the DLN compared to LCMV, with RRV localizing principally to the subcapsular region and LCMV to the paracortical T-cell zones. Single-cell RNA-sequencing analysis of adoptively transferred gp33-specific transgenic CD8+ T cells showed rapid differentiation into effector cells after LCMV but not RRV infection. This defect in RRV-specific CD8+ T effector cell maturation was corrected by local blockade of type I interferon (IFN) signaling, which also resulted in increased RRV infection in the DLN. Studies in Wdfy4-/-, CD11c-Cre B2mfl/fl, or Xcr1-Cre Ifnar1fl/fl mice that respectively lack cross-presenting capacity, MHC-I antigen presentation by dendritic cells (DCs), or type I IFN signaling in the DC1 subset show that RRV-specific CD8+ T-cell responses can be improved by enhanced direct antigen presentation by DCs. Overall, our experiments suggest that antiviral type I IFN signaling in DCs limits direct alphavirus infection and antigen presentation, which likely delays CD8+ T-cell responses.IMPORTANCEChronic arthritis and musculoskeletal disease are common outcomes of infections caused by arthritogenic alphaviruses, including Ross River virus (RRV), due to incomplete virus clearance. Unlike other viral infections that are efficiently cleared by cytotoxic CD8+ T cells, RRV infection is surprisingly unaffected by CD8+ T cells as mice lacking or having these cells show similar viral persistence in joint and lymphoid tissues. To elucidate the basis for this deficient response, we measured the RRV-specific CD8+ T-cell population size and activation state relative to another virus known to elicit a strong T-cell response. Our findings reveal that RRV induces fewer CD8+ T cells due to limited infection of immune cells in the draining lymph node. By increasing RRV susceptibility in antigen-presenting cells, we elicited a robust CD8+ T-cell response. These results highlight antigen availability and virus tropism as possible targets for intervention against RRV immune evasion and persistence.
罗斯河病毒(RRV)和其他α-病毒会导致慢性肌肉骨骼综合征,这些综合征与病毒持续存在有关,这表明免疫清除机制(包括 CD8+ T 细胞反应)存在缺陷。在这里,我们使用了表达淋巴细胞性脉络膜炎病毒(LCMV)免疫优势 CD8+ T 细胞表位的重组 RRV-gp33,将其与可强烈诱导抗病毒 CD8+ T 细胞的 LCMV 病毒的反应进行了直接比较。脚垫注射 RRV-gp33 后,我们在引流淋巴结(DLN)中检测到的 gp33 特异性 CD8+ T 细胞少于 LCMV 感染,尽管脚部的病毒 RNA 水平相似。然而,与 LCMV 相比,在 DLN 中检测到的 RRV RNA 更少,RRV 主要定位于囊下区,而 LCMV 则定位于皮质旁 T 细胞区。对收养转移的 gp33 特异性转基因 CD8+ T 细胞进行的单细胞 RNA 序列分析表明,LCMV 感染后,CD8+ T 细胞迅速分化为效应细胞,而 RRV 感染后则不然。这种 RRV 特异性 CD8+ T 效应细胞成熟的缺陷可通过局部阻断 I 型干扰素 (IFN) 信号转导得到纠正,这也导致了 DLN 中 RRV 感染的增加。在Wdfy4-/-、CD11c-Cre B2mfl/fl或Xcr1-Cre Ifnar1fl/fl小鼠中进行的研究表明,RRV特异性CD8+ T细胞反应可以通过增强DC的直接抗原呈递得到改善。总之,我们的实验表明,DC 中的 I 型 IFN 抗病毒信号限制了α-病毒的直接感染和抗原递呈,这可能会延迟 CD8+ T 细胞应答。与其他能被细胞毒性 CD8+ T 细胞有效清除的病毒感染不同,RRV 感染出人意料地不受 CD8+ T 细胞的影响,因为缺乏或拥有这些细胞的小鼠在关节和淋巴组织中表现出类似的病毒持久性。为了阐明这种反应缺陷的基础,我们测量了 RRV 特异性 CD8+ T 细胞群的大小和活化状态,并与另一种已知能引起强烈 T 细胞反应的病毒进行了比较。我们的研究结果表明,由于引流淋巴结中的免疫细胞感染有限,RRV 诱导的 CD8+ T 细胞数量较少。通过提高抗原递呈细胞对 RRV 的敏感性,我们诱发了强大的 CD8+ T 细胞反应。这些结果突出表明,抗原可用性和病毒趋向性是干预 RRV 免疫逃避和持续存在的可能目标。
{"title":"Type I interferon signaling in dendritic cells limits direct antigen presentation and CD8<sup>+</sup> T cell responses against an arthritogenic alphavirus.","authors":"Christopher B Bullock, Leran Wang, Brian C Ware, Ngan Wagoner, Ray A Ohara, Tian-Tian Liu, Pritesh Desai, Bjoern Peters, Kenneth M Murphy, Scott A Handley, Thomas E Morrison, Michael S Diamond","doi":"10.1128/mbio.02930-24","DOIUrl":"https://doi.org/10.1128/mbio.02930-24","url":null,"abstract":"<p><p>Ross River virus (RRV) and other alphaviruses cause chronic musculoskeletal syndromes that are associated with viral persistence, which suggests deficits in immune clearance mechanisms, including CD8<sup>+</sup> T-cell responses. Here, we used a recombinant RRV-gp33 that expresses the immunodominant CD8<sup>+</sup> T-cell epitope of lymphocytic choriomeningitis virus (LCMV) to directly compare responses with a virus, LCMV, that strongly induces antiviral CD8<sup>+</sup> T cells. After footpad injection, we detected fewer gp33-specific CD8<sup>+</sup> T cells in the draining lymph node (DLN) after RRV-gp33 than LCMV infection, despite similar viral RNA levels in the foot. However, less RRV RNA was detected in the DLN compared to LCMV, with RRV localizing principally to the subcapsular region and LCMV to the paracortical T-cell zones. Single-cell RNA-sequencing analysis of adoptively transferred gp33-specific transgenic CD8<sup>+</sup> T cells showed rapid differentiation into effector cells after LCMV but not RRV infection. This defect in RRV-specific CD8<sup>+</sup> T effector cell maturation was corrected by local blockade of type I interferon (IFN) signaling, which also resulted in increased RRV infection in the DLN. Studies in <i>Wdfy4</i><sup>-/-</sup>, CD11c-Cre <i>B2m</i><sup>fl/fl</sup>, or Xcr1-Cre <i>Ifnar1</i><sup>fl/fl</sup> mice that respectively lack cross-presenting capacity, MHC-I antigen presentation by dendritic cells (DCs), or type I IFN signaling in the DC1 subset show that RRV-specific CD8<sup>+</sup> T-cell responses can be improved by enhanced direct antigen presentation by DCs. Overall, our experiments suggest that antiviral type I IFN signaling in DCs limits direct alphavirus infection and antigen presentation, which likely delays CD8<sup>+</sup> T-cell responses.IMPORTANCEChronic arthritis and musculoskeletal disease are common outcomes of infections caused by arthritogenic alphaviruses, including Ross River virus (RRV), due to incomplete virus clearance. Unlike other viral infections that are efficiently cleared by cytotoxic CD8<sup>+</sup> T cells, RRV infection is surprisingly unaffected by CD8<sup>+</sup> T cells as mice lacking or having these cells show similar viral persistence in joint and lymphoid tissues. To elucidate the basis for this deficient response, we measured the RRV-specific CD8<sup>+</sup> T-cell population size and activation state relative to another virus known to elicit a strong T-cell response. Our findings reveal that RRV induces fewer CD8<sup>+</sup> T cells due to limited infection of immune cells in the draining lymph node. By increasing RRV susceptibility in antigen-presenting cells, we elicited a robust CD8<sup>+</sup> T-cell response. These results highlight antigen availability and virus tropism as possible targets for intervention against RRV immune evasion and persistence.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0293024"},"PeriodicalIF":5.1,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142622369","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Excessive inflammation upon Chlamydia trachomatis infection can cause severe damages in the female genital tract. This obligate intracellular bacterium develops mainly in epithelial cells, whose innate response contributes to the overall inflammatory response to infection. The ubiquitin-like protein interferon-stimulated gene 15 (ISG15) stimulates interferon γ (IFNγ) production and is required for bacterial clearance in several infectious contexts. Here, we describe and investigate the consequences of the increase in ISG15 expression by epithelial cells infected with C. trachomatis. Infection of HeLa cells and primary ecto-cervical epithelial cells resulted in a transcriptional upregulation of ISG15 expression. This did not involve the canonical type I interferon (IFN-I) signaling pathway and depended instead on the activation of the STING/TBK1/IRF3 pathway. The absence or reduction of ISG15 synthesis led to increased production of several cytokines and chemokines, including interleukin (IL) 6 and IL8. This implicates that ISG15 normally dampens the immune response induced by C. trachomatis infection in epithelial cells. ISG15 exerted its control from an intracellular location, but without involving ISGylation. Finally, higher levels of inflammation and delayed bacterial clearance were observed in the genital tracts of ISG15-KO mice infected by C. trachomatis compared with wild-type animals; however, IFNγ production was unchanged. Altogether, our data show that ISG15 expression acts as a brake on the immune response to C. trachomatis infection in epithelial cells and limits bacterial burden and inflammation in mice.IMPORTANCEInfection of epithelial cells by Chlamydia trachomatis elicits an innate immune response by these cells. The signaling pathways involved, and their outcomes, are still very poorly understood. In this paper, we described how Chlamydia infection triggered the expression of ISG15, a small molecule normally associated to type I interferon (IFN-I) signaling and control of INF-γ production. ISG15 synthesis by epithelial cells attenuated their immune response to Chlamydia infection. In mice, we observed that ISG15 displayed a marginal role in modulating the production of IFN-γ, a key component of the host immune response to infection, but facilitated bacterial clearance. Overall, our study strengthens the importance of ISG15 not only in the resolution of viral but also of bacterial infection and document its role of "immune brake" in the context of Chlamydia infection.
{"title":"<i>Chlamydia</i>-driven ISG15 expression dampens the immune response of epithelial cells independently of ISGylation.","authors":"Yongzheng Wu, Chang Liu, Chongfa Tang, Béatrice Niragire, Yaël Levy-Zauberman, Cindy Adapen, Thomas Vernay, Juliette Hugueny, Véronique Baud, Agathe Subtil","doi":"10.1128/mbio.02401-24","DOIUrl":"10.1128/mbio.02401-24","url":null,"abstract":"<p><p>Excessive inflammation upon <i>Chlamydia trachomatis</i> infection can cause severe damages in the female genital tract. This obligate intracellular bacterium develops mainly in epithelial cells, whose innate response contributes to the overall inflammatory response to infection. The ubiquitin-like protein interferon-stimulated gene 15 (ISG15) stimulates interferon γ (IFNγ) production and is required for bacterial clearance in several infectious contexts. Here, we describe and investigate the consequences of the increase in ISG15 expression by epithelial cells infected with <i>C. trachomatis</i>. Infection of HeLa cells and primary ecto-cervical epithelial cells resulted in a transcriptional upregulation of <i>ISG15</i> expression. This did not involve the canonical type I interferon (IFN-I) signaling pathway and depended instead on the activation of the STING/TBK1/IRF3 pathway. The absence or reduction of ISG15 synthesis led to increased production of several cytokines and chemokines, including interleukin (IL) 6 and IL8. This implicates that ISG15 normally dampens the immune response induced by <i>C. trachomatis</i> infection in epithelial cells. ISG15 exerted its control from an intracellular location, but without involving ISGylation. Finally, higher levels of inflammation and delayed bacterial clearance were observed in the genital tracts of ISG15-KO mice infected by <i>C. trachomatis</i> compared with wild-type animals; however, IFNγ production was unchanged. Altogether, our data show that ISG15 expression acts as a brake on the immune response to <i>C. trachomatis</i> infection in epithelial cells and limits bacterial burden and inflammation in mice.IMPORTANCEInfection of epithelial cells by <i>Chlamydia trachomatis</i> elicits an innate immune response by these cells. The signaling pathways involved, and their outcomes, are still very poorly understood. In this paper, we described how <i>Chlamydia</i> infection triggered the expression of ISG15, a small molecule normally associated to type I interferon (IFN-I) signaling and control of INF-γ production. ISG15 synthesis by epithelial cells attenuated their immune response to <i>Chlamydia</i> infection. In mice, we observed that ISG15 displayed a marginal role in modulating the production of IFN-γ, a key component of the host immune response to infection, but facilitated bacterial clearance. Overall, our study strengthens the importance of ISG15 not only in the resolution of viral but also of bacterial infection and document its role of \"immune brake\" in the context of <i>Chlamydia</i> infection.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0240124"},"PeriodicalIF":5.1,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11559041/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142349634","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-13Epub Date: 2024-09-30DOI: 10.1128/mbio.02485-24
Annesha Ghosh, Jogender Singh
Diverse microbial pathogens are known to attenuate host protein synthesis. Consequently, the host mounts a defense response against protein translation inhibition, leading to increased transcript levels of immune genes. The seemingly paradoxical upregulation of immune gene transcripts in response to blocked protein synthesis suggests that the defense mechanism against translation inhibition may not universally benefit host survival. However, a comprehensive assessment of host survival on pathogens upon blockage of different stages of protein synthesis is currently lacking. Here, we investigate the impact of knockdown of various translation initiation and elongation factors on the survival of Caenorhabditis elegans exposed to Pseudomonas aeruginosa. Intriguingly, we observe opposing effects on C. elegans survival depending on whether translation initiation or elongation is inhibited. While translation initiation inhibition enhances survival, elongation inhibition decreases it. Transcriptomic studies reveal that translation initiation inhibition activates a bZIP transcription factor ZIP-2-dependent innate immune response that protects C. elegans from P. aeruginosa infection. In contrast, inhibiting translation elongation triggers both ZIP-2-dependent and ZIP-2-independent immune responses that, while effective in clearing the infection, are detrimental to the host. Thus, our findings reveal the opposing roles of translation initiation and elongation inhibition in C. elegans survival during P. aeruginosa infection, highlighting distinct transcriptional reprogramming that may underlie these differences.
Importance: Several microbial pathogens target host protein synthesis machinery, potentially limiting the innate immune responses of the host. In response, hosts trigger a defensive response, elevating immune gene transcripts. This counterintuitive response can have either beneficial or harmful effects on host survival. In this study, we conduct a comprehensive analysis of the impact of knocking down various translation initiation and elongation factors on the survival of Caenorhabditis elegans exposed to Pseudomonas aeruginosa. Intriguingly, inhibiting initiation and elongation factors has contrasting effects on C. elegans survival. Inhibiting translation initiation activates immune responses that protect the host from bacterial infection, while inhibiting translation elongation induces aberrant immune responses that, although clear the infection, are detrimental to the host. Our study reveals divergent roles of translation initiation and elongation inhibition in C. elegans survival during P. aeruginosa infection and identifies differential transcriptional reprogramming that could underlie these differences.
{"title":"Translation initiation or elongation inhibition triggers contrasting effects on <i>Caenorhabditis elegans</i> survival during pathogen infection.","authors":"Annesha Ghosh, Jogender Singh","doi":"10.1128/mbio.02485-24","DOIUrl":"10.1128/mbio.02485-24","url":null,"abstract":"<p><p>Diverse microbial pathogens are known to attenuate host protein synthesis. Consequently, the host mounts a defense response against protein translation inhibition, leading to increased transcript levels of immune genes. The seemingly paradoxical upregulation of immune gene transcripts in response to blocked protein synthesis suggests that the defense mechanism against translation inhibition may not universally benefit host survival. However, a comprehensive assessment of host survival on pathogens upon blockage of different stages of protein synthesis is currently lacking. Here, we investigate the impact of knockdown of various translation initiation and elongation factors on the survival of <i>Caenorhabditis elegans</i> exposed to <i>Pseudomonas aeruginosa</i>. Intriguingly, we observe opposing effects on <i>C. elegans</i> survival depending on whether translation initiation or elongation is inhibited. While translation initiation inhibition enhances survival, elongation inhibition decreases it. Transcriptomic studies reveal that translation initiation inhibition activates a bZIP transcription factor ZIP-2-dependent innate immune response that protects <i>C. elegans</i> from <i>P. aeruginosa</i> infection. In contrast, inhibiting translation elongation triggers both ZIP-2-dependent and ZIP-2-independent immune responses that, while effective in clearing the infection, are detrimental to the host. Thus, our findings reveal the opposing roles of translation initiation and elongation inhibition in <i>C. elegans</i> survival during <i>P. aeruginosa</i> infection, highlighting distinct transcriptional reprogramming that may underlie these differences.</p><p><strong>Importance: </strong>Several microbial pathogens target host protein synthesis machinery, potentially limiting the innate immune responses of the host. In response, hosts trigger a defensive response, elevating immune gene transcripts. This counterintuitive response can have either beneficial or harmful effects on host survival. In this study, we conduct a comprehensive analysis of the impact of knocking down various translation initiation and elongation factors on the survival of <i>Caenorhabditis elegans</i> exposed to <i>Pseudomonas aeruginosa</i>. Intriguingly, inhibiting initiation and elongation factors has contrasting effects on <i>C. elegans</i> survival. Inhibiting translation initiation activates immune responses that protect the host from bacterial infection, while inhibiting translation elongation induces aberrant immune responses that, although clear the infection, are detrimental to the host. Our study reveals divergent roles of translation initiation and elongation inhibition in <i>C. elegans</i> survival during <i>P. aeruginosa</i> infection and identifies differential transcriptional reprogramming that could underlie these differences.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0248524"},"PeriodicalIF":5.1,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11559039/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142349646","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-13Epub Date: 2024-10-09DOI: 10.1128/mbio.02327-24
Kyle Stearns, George Lampe, Rachel Hanan, Tara Marcink, Stefan Niewiesk, Samuel H Sternberg, Alexander L Greninger, Matteo Porotto, Anne Moscona
Human parainfluenza virus 3 (HPIV3) infection is driven by the coordinated action of viral surface glycoproteins hemagglutinin-neuraminidase (HN) and fusion protein (F). Receptor-engaged HN activates F to insert into the target cell membrane and drive virion-cell membrane fusion. For F to mediate entry, its precursor (F0) must first be cleaved by host proteases. F0 cleavage has been thought to be executed during viral glycoprotein transit through the trans-Golgi network by the ubiquitously expressed furin because F0 proteins of laboratory-adapted viruses contain a furin recognition dibasic cleavage motif RXKR around residue 108. Here, we show that the F proteins of field strains have a different cleavage motif from laboratory-adapted strains and are cleaved by unidentified proteases expressed in only a narrow subset of cell types. We demonstrate that extracellular serine protease inhibitors block HPIV3 F0 cleavage for field strains, suggesting F0 cleavage occurs at the cell surface facilitated by transmembrane proteases. Candidate proteases that may process HPIV3 F in vivo were identified by a genome-wide CRISPRa screen in HEK293/dCas9-VP64 + MPH cells. The lung-expressed extracellular serine proteases TMPRSS2 and TMPRSS13 are both sufficient to cleave HPIV3 F and enable infectious virus release by otherwise non-permissive cells. Our findings support an alternative mechanism of F activation in vivo, reliant on extracellular membrane-bound serine proteases expressed in a narrow subset of cells. The proportion of HPIV3 F proteins cleaved and infectious virus release is determined by host cell expression of requisite proteases, allowing just-in-time activation of F and positioning F cleavage as another key regulator of HPIV3 spread.
Importance: Enveloped viruses cause a wide range of diseases in humans. At the first step of infection, these viruses must fuse their envelope with a cell membrane to initiate infection. This fusion is mediated by viral proteins that require a critical activating cleavage event. It was previously thought that for parainfluenza virus 3, an important cause of respiratory disease and a representative of a group of important pathogens, this cleavage event was mediated by furin in the cell secretory pathways prior to formation of the virions. We show that this is only true for laboratory strain viruses, and that clinical viruses that infect humans utilize extracellular proteases that are only made by a small subset of cells. These results highlight the importance of studying authentic clinical viruses that infect human tissues for understanding natural infection.
人副流感病毒 3(HPIV3)感染是由病毒表面糖蛋白血凝素-神经氨酸酶(HN)和融合蛋白(F)的协调作用驱动的。受体激活的 HN 可激活 F,使其插入靶细胞膜并驱动病毒细胞膜融合。F 的前体(F0)必须首先被宿主蛋白酶裂解,才能介导 F 进入。F0 的裂解被认为是在病毒糖蛋白通过转高尔基网络的过程中由普遍表达的呋喃蛋白完成的,因为实验室适应病毒的 F0 蛋白含有一个呋喃蛋白识别二碱基裂解基序 RXKR,该基序位于残基 108 附近。在这里,我们发现野外毒株的 F 蛋白具有不同于实验室适应性毒株的裂解基序,并且只被在少数细胞类型中表达的不明蛋白酶裂解。我们证明,细胞外丝氨酸蛋白酶抑制剂阻断了野外菌株的 HPIV3 F0 裂解,这表明 F0 裂解发生在细胞表面,由跨膜蛋白酶促进。通过在 HEK293/dCas9-VP64 + MPH 细胞中进行全基因组 CRISPRa 筛选,确定了可能在体内处理 HPIV3 F 的候选蛋白酶。肺部表达的细胞外丝氨酸蛋白酶TMPRSS2和TMPRSS13都足以裂解HPIV3 F,并使原本不允许感染的细胞释放出传染性病毒。我们的研究结果支持体内 F 激活的另一种机制,即依赖于在少数细胞亚群中表达的胞外膜结合丝氨酸蛋白酶。HPIV3 F蛋白被裂解的比例和传染性病毒的释放由宿主细胞表达的必要蛋白酶决定,这使得F能够及时激活,并将F裂解定位为HPIV3传播的另一个关键调节因子:包膜病毒可导致人类多种疾病。在感染的第一步,这些病毒必须将其包膜与细胞膜融合,以启动感染。这种融合是由病毒蛋白介导的,需要一个关键的激活裂解事件。以前人们认为,对于副流感病毒 3(呼吸道疾病的重要病因和一组重要病原体的代表)来说,这种裂解事件是在病毒形成之前由细胞分泌途径中的呋喃介导的。我们的研究表明,这只适用于实验室毒株病毒,而感染人类的临床病毒利用的胞外蛋白酶只由一小部分细胞制造。这些结果凸显了研究感染人体组织的真实临床病毒对于了解自然感染的重要性。
{"title":"Human parainfluenza virus 3 field strains undergo extracellular fusion protein cleavage to activate entry.","authors":"Kyle Stearns, George Lampe, Rachel Hanan, Tara Marcink, Stefan Niewiesk, Samuel H Sternberg, Alexander L Greninger, Matteo Porotto, Anne Moscona","doi":"10.1128/mbio.02327-24","DOIUrl":"10.1128/mbio.02327-24","url":null,"abstract":"<p><p>Human parainfluenza virus 3 (HPIV3) infection is driven by the coordinated action of viral surface glycoproteins hemagglutinin-neuraminidase (HN) and fusion protein (F). Receptor-engaged HN activates F to insert into the target cell membrane and drive virion-cell membrane fusion. For F to mediate entry, its precursor (F0) must first be cleaved by host proteases. F0 cleavage has been thought to be executed during viral glycoprotein transit through the trans-Golgi network by the ubiquitously expressed furin because F0 proteins of laboratory-adapted viruses contain a furin recognition dibasic cleavage motif RXKR around residue 108. Here, we show that the F proteins of field strains have a different cleavage motif from laboratory-adapted strains and are cleaved by unidentified proteases expressed in only a narrow subset of cell types. We demonstrate that extracellular serine protease inhibitors block HPIV3 F0 cleavage for field strains, suggesting F0 cleavage occurs at the cell surface facilitated by transmembrane proteases. Candidate proteases that may process HPIV3 F <i>in vivo</i> were identified by a genome-wide CRISPRa screen in HEK293/dCas9-VP64 + MPH cells. The lung-expressed extracellular serine proteases TMPRSS2 and TMPRSS13 are both sufficient to cleave HPIV3 F and enable infectious virus release by otherwise non-permissive cells. Our findings support an alternative mechanism of F activation <i>in vivo</i>, reliant on extracellular membrane-bound serine proteases expressed in a narrow subset of cells. The proportion of HPIV3 F proteins cleaved and infectious virus release is determined by host cell expression of requisite proteases, allowing just-in-time activation of F and positioning F cleavage as another key regulator of HPIV3 spread.</p><p><strong>Importance: </strong>Enveloped viruses cause a wide range of diseases in humans. At the first step of infection, these viruses must fuse their envelope with a cell membrane to initiate infection. This fusion is mediated by viral proteins that require a critical activating cleavage event. It was previously thought that for parainfluenza virus 3, an important cause of respiratory disease and a representative of a group of important pathogens, this cleavage event was mediated by furin in the cell secretory pathways prior to formation of the virions. We show that this is only true for laboratory strain viruses, and that clinical viruses that infect humans utilize extracellular proteases that are only made by a small subset of cells. These results highlight the importance of studying authentic clinical viruses that infect human tissues for understanding natural infection.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0232724"},"PeriodicalIF":5.1,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11559058/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142391670","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}