Lauren J Gonsalves, Allyson Tran, Tessa Gardiner, Tiia Freeman, Angshita Dutta, Carson J Miller, Sharon McNamara, Adam Waalkes, Dustin R Long, Stephen J Salipante, Lucas R Hoffman, Daniel J Wolter
Trimethoprim-sulfamethoxazole (SXT) is commonly used to treat diverse Staphylococcus aureus infections, including those associated with cystic fibrosis (CF) pulmonary disease. Studies with Escherichia coli found that SXT impairs tetrahydrofolate production, leading to DNA damage, stress response induction, and accumulation of reactive oxygen species (ROS) in a process known as thymineless death (TLD). TLD survival can occur through the uptake of exogenous thymidine, countering the effects of SXT; however, a growing body of research has implicated central metabolism as another potentially important determinant of bacterial survival of SXT and other antibiotics. Here, we conducted studies to better understand the mechanisms of TLD survival in S. aureus. We found that thymidine abundances in CF sputum were insufficient to prevent TLD of S. aureus, highlighting the importance of alternative survival mechanisms in vivo. In S. aureus cultured in vitro with SXT and low thymidine, we frequently identified adaptive mutations in genes encoding carbohydrate, nucleotide, and amino acid metabolism, supporting reduced metabolism as a common survival mechanism. Although intracellular ROS levels rose with SXT treatment in vitro, survival was not improved in the presence of ROS scavengers, unlike in E. coli. SXT challenge induced the SOS response, which was alleviated by added thymidine. Finally, an inactivating mutation in the phosphotransferase gene ptsI conferred both limitation in cellular ATP and improved survival against TLD. Collectively, these results suggest that alterations in core metabolic functions, particularly those that reduce ATP levels, predominantly confer S. aureus survival and persistence during SXT treatment, potentially identifying novel targets for co-treatment.IMPORTANCEStaphylococcus aureus is a ubiquitous organism and one of the leading causes of human infections, many of which are difficult to treat due to persistence, antibiotic resistance, or antibiotic tolerance. As our arsenal of effective antibiotics dwindles, the need for improved treatments becomes increasingly urgent, necessitating a better understanding of the precise mechanisms by which pathogens evade our most critical antimicrobial agents. Here, we report a systematic characterization of the mechanisms of S. aureus survival to treatment with the first-line antistaphylococcal antibiotic trimethoprim-sulfamethoxazole, identifying pathways and candidate targets for enhancing the efficacy of available antimicrobial agents.
{"title":"Mechanisms of <i>Staphylococcus aureus</i> survival of trimethoprim-sulfamethoxazole-induced thymineless death.","authors":"Lauren J Gonsalves, Allyson Tran, Tessa Gardiner, Tiia Freeman, Angshita Dutta, Carson J Miller, Sharon McNamara, Adam Waalkes, Dustin R Long, Stephen J Salipante, Lucas R Hoffman, Daniel J Wolter","doi":"10.1128/mbio.01634-24","DOIUrl":"https://doi.org/10.1128/mbio.01634-24","url":null,"abstract":"<p><p>Trimethoprim-sulfamethoxazole (SXT) is commonly used to treat diverse <i>Staphylococcus aureus</i> infections, including those associated with cystic fibrosis (CF) pulmonary disease. Studies with <i>Escherichia coli</i> found that SXT impairs tetrahydrofolate production, leading to DNA damage, stress response induction, and accumulation of reactive oxygen species (ROS) in a process known as thymineless death (TLD). TLD survival can occur through the uptake of exogenous thymidine, countering the effects of SXT; however, a growing body of research has implicated central metabolism as another potentially important determinant of bacterial survival of SXT and other antibiotics. Here, we conducted studies to better understand the mechanisms of TLD survival in <i>S. aureus</i>. We found that thymidine abundances in CF sputum were insufficient to prevent TLD of <i>S. aureus</i>, highlighting the importance of alternative survival mechanisms <i>in vivo</i>. In <i>S. aureus</i> cultured <i>in vitro</i> with SXT and low thymidine, we frequently identified adaptive mutations in genes encoding carbohydrate, nucleotide, and amino acid metabolism, supporting reduced metabolism as a common survival mechanism. Although intracellular ROS levels rose with SXT treatment <i>in vitro</i>, survival was not improved in the presence of ROS scavengers, unlike in <i>E. coli</i>. SXT challenge induced the SOS response, which was alleviated by added thymidine. Finally, an inactivating mutation in the phosphotransferase gene <i>ptsI</i> conferred both limitation in cellular ATP and improved survival against TLD. Collectively, these results suggest that alterations in core metabolic functions, particularly those that reduce ATP levels, predominantly confer <i>S. aureus</i> survival and persistence during SXT treatment, potentially identifying novel targets for co-treatment.IMPORTANCE<i>Staphylococcus aureus</i> is a ubiquitous organism and one of the leading causes of human infections, many of which are difficult to treat due to persistence, antibiotic resistance, or antibiotic tolerance. As our arsenal of effective antibiotics dwindles, the need for improved treatments becomes increasingly urgent, necessitating a better understanding of the precise mechanisms by which pathogens evade our most critical antimicrobial agents. Here, we report a systematic characterization of the mechanisms of <i>S. aureus</i> survival to treatment with the first-line antistaphylococcal antibiotic trimethoprim-sulfamethoxazole, identifying pathways and candidate targets for enhancing the efficacy of available antimicrobial agents.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142503525","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}
Joshua Soto Ocaña, Elliot S Friedman, Orlaith Keenan, Nile U Bayard, Eileen Ford, Ceylan Tanes, Matthew J Munneke, William N Beavers, Eric P Skaar, Kyle Bittinger, Babette S Zemel, Gary D Wu, Joseph P Zackular
The gut microbiota plays a critical role in human health and disease. Microbial community assembly and succession early in life are influenced by numerous factors. In turn, assembly of this microbial community is known to influence the host, including immune system development, and has been linked to outcomes later in life. To date, the role of host-mediated nutritional immunity and metal availability in shaping microbial community assembly and succession early in life has not been explored in depth. Using a human infant cohort, we show that the metal-chelating protein calprotectin is highly abundant in infants. Taxa previously shown to be successful early colonizers of the infant gut, such as Enterococcus, Enterobacteriaceae, and Bacteroides, are highly resistant to experimental metal starvation in culture. Lactobacillus, meanwhile, is highly susceptible to metal restriction, pointing to a possible mechanism by which host-mediated metal limitation shapes the fitness of early colonizing taxa in the infant gut. We further demonstrate that formula-fed infants harbor markedly higher levels of metals in their gastrointestinal tract compared to breastfed infants. Formula-fed infants with high levels of metals harbor distinct microbial communities compared to breastfed infants, with higher levels of Enterococcus, Enterobacter, and Klebsiella, taxa which show increased resistance to the toxic effects of high metal concentrations. These data highlight a new paradigm in microbial community assembly and suggest an unappreciated role for nutritional immunity and dietary metals in shaping the earliest colonization events of the microbiota.IMPORTANCEEarly life represents a critical window for microbial colonization of the human gastrointestinal tract. Surprisingly, we still know little about the rules that govern the successful colonization of infants and the factors that shape the success of early life microbial colonizers. In this study, we report that metal availability is an important factor in the assembly and succession of the early life microbiota. We show that the host-derived metal-chelating protein, calprotectin, is highly abundant in infants and successful early life colonizers can overcome metal restriction. We further demonstrate that feeding modality (breastmilk vs formula) markedly impacts metal levels in the gut, potentially influencing microbial community succession. Our work suggests that metals, a previously unexplored aspect of early life ecology, may play a critical role in shaping the early events of microbiota assembly in infants.
{"title":"Metal availability shapes early life microbial ecology and community succession.","authors":"Joshua Soto Ocaña, Elliot S Friedman, Orlaith Keenan, Nile U Bayard, Eileen Ford, Ceylan Tanes, Matthew J Munneke, William N Beavers, Eric P Skaar, Kyle Bittinger, Babette S Zemel, Gary D Wu, Joseph P Zackular","doi":"10.1128/mbio.01534-24","DOIUrl":"https://doi.org/10.1128/mbio.01534-24","url":null,"abstract":"<p><p>The gut microbiota plays a critical role in human health and disease. Microbial community assembly and succession early in life are influenced by numerous factors. In turn, assembly of this microbial community is known to influence the host, including immune system development, and has been linked to outcomes later in life. To date, the role of host-mediated nutritional immunity and metal availability in shaping microbial community assembly and succession early in life has not been explored in depth. Using a human infant cohort, we show that the metal-chelating protein calprotectin is highly abundant in infants. Taxa previously shown to be successful early colonizers of the infant gut, such as <i>Enterococcus</i>, <i>Enterobacteriaceae,</i> and <i>Bacteroides,</i> are highly resistant to experimental metal starvation in culture. <i>Lactobacillus</i>, meanwhile, is highly susceptible to metal restriction, pointing to a possible mechanism by which host-mediated metal limitation shapes the fitness of early colonizing taxa in the infant gut. We further demonstrate that formula-fed infants harbor markedly higher levels of metals in their gastrointestinal tract compared to breastfed infants. Formula-fed infants with high levels of metals harbor distinct microbial communities compared to breastfed infants, with higher levels of <i>Enterococcus</i>, <i>Enterobacter,</i> and <i>Klebsiella</i>, taxa which show increased resistance to the toxic effects of high metal concentrations. These data highlight a new paradigm in microbial community assembly and suggest an unappreciated role for nutritional immunity and dietary metals in shaping the earliest colonization events of the microbiota.IMPORTANCEEarly life represents a critical window for microbial colonization of the human gastrointestinal tract. Surprisingly, we still know little about the rules that govern the successful colonization of infants and the factors that shape the success of early life microbial colonizers. In this study, we report that metal availability is an important factor in the assembly and succession of the early life microbiota. We show that the host-derived metal-chelating protein, calprotectin, is highly abundant in infants and successful early life colonizers can overcome metal restriction. We further demonstrate that feeding modality (breastmilk vs formula) markedly impacts metal levels in the gut, potentially influencing microbial community succession. Our work suggests that metals, a previously unexplored aspect of early life ecology, may play a critical role in shaping the early events of microbiota assembly in infants.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142503526","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}
Though a confined or a broad population is exposed respectively to endemic or pandemic infections, in the same environment, some individuals resist the development of infections. The attributed reason is the inheritance of a set of immune system genes that can efficiently deal with the pathogens. In this study, we show how outbred mice differentially respond to Cryptococcus neoformans, a fungal pathogen, and the mechanism through which the surviving mice mount a protective immune defense. We identified that those mice developing antibodies specifically against Pep1p, an aspartic protease secreted by C. neoformans, had significantly improved survival. Vaccination (either prophylactic or therapeutic) with a recombinant Pep1p significantly increased the survival of the mice by decreasing the fungal load and stimulating a protective immune response. Passive immunization of C. neoformans-infected mice with monoclonal antibodies developed against Pep1p also improves the survival of the mice by increasing phagocytosis of C. neoformans and decreasing the multiplication of this fungus. Together, these data demonstrate the prophylactic and therapeutic potentials of the C. neoformans antigenic protein Pep1p or Pep1p-specific antibodies against this fungal infection. Also, this study suggests that the immunological interaction and thereby the responses developed against a pathogen guide the hosts to behave differentially against microbial pathogenicity.
Importance: Vaccination and immunotherapies against fungal pathogens still remain a challenge. Here, we show using an in vivo model based on outbred mice that development of antibodies against Pep1p, an antigenic protein of the fungal pathogen Cryptococcus neoformans, confers resistance to this fungal infection. In support of this observation, prophylactic or therapeutic immunization of the mice with recombinant Pep1p could improve their survival when infected with a lethal dose of C. neoformans. Moreover, passive therapy with monoclonal anti-Pep1p antibodies also enhanced survival of the mice from C. neoformans infection. The associated antifungal mechanisms were mounting of a protective immune response and the development of fungal specific antibodies that decrease the fungal burden due to an increase in their phagocytosis and/or inhibit the fungal multiplication. Together, our study demonstrates (a) the mode of host-fungal interaction and the immune response developed thereby play a crucial role in developing resistance against C. neoformans; (b) Pep1p, an aspartic protease as well as an antigenic protein secreted by C. neoformans, can be exploited for vaccination (both prophylactic and therapeutic) or immunotherapy to improve the host defense during this fungal infection.
{"title":"<i>Cryptococcus neoformans</i> infections: aspartyl protease potential to improve outcome in susceptible hosts.","authors":"Frédérique Vernel-Pauillac, Christine Laurent-Winter, Laurence Fiette, Guilhem Janbon, Vishukumar Aimanianda, Françoise Dromer","doi":"10.1128/mbio.02733-24","DOIUrl":"https://doi.org/10.1128/mbio.02733-24","url":null,"abstract":"<p><p>Though a confined or a broad population is exposed respectively to endemic or pandemic infections, in the same environment, some individuals resist the development of infections. The attributed reason is the inheritance of a set of immune system genes that can efficiently deal with the pathogens. In this study, we show how outbred mice differentially respond to <i>Cryptococcus neoformans,</i> a fungal pathogen, and the mechanism through which the surviving mice mount a protective immune defense. We identified that those mice developing antibodies specifically against Pep1p, an aspartic protease secreted by <i>C. neoformans</i>, had significantly improved survival. Vaccination (either prophylactic or therapeutic) with a recombinant Pep1p significantly increased the survival of the mice by decreasing the fungal load and stimulating a protective immune response. Passive immunization of <i>C. neoformans-</i>infected mice with monoclonal antibodies developed against Pep1p also improves the survival of the mice by increasing phagocytosis of <i>C. neoformans</i> and decreasing the multiplication of this fungus. Together, these data demonstrate the prophylactic and therapeutic potentials of the <i>C. neoformans</i> antigenic protein Pep1p or Pep1p-specific antibodies against this fungal infection. Also, this study suggests that the immunological interaction and thereby the responses developed against a pathogen guide the hosts to behave differentially against microbial pathogenicity.</p><p><strong>Importance: </strong>Vaccination and immunotherapies against fungal pathogens still remain a challenge. Here, we show using an <i>in vivo</i> model based on outbred mice that development of antibodies against Pep1p, an antigenic protein of the fungal pathogen <i>Cryptococcus neoformans</i>, confers resistance to this fungal infection. In support of this observation, prophylactic or therapeutic immunization of the mice with recombinant Pep1p could improve their survival when infected with a lethal dose of <i>C. neoformans</i>. Moreover, passive therapy with monoclonal anti-Pep1p antibodies also enhanced survival of the mice from <i>C. neoformans</i> infection. The associated antifungal mechanisms were mounting of a protective immune response and the development of fungal specific antibodies that decrease the fungal burden due to an increase in their phagocytosis and/or inhibit the fungal multiplication. Together, our study demonstrates (a) the mode of host-fungal interaction and the immune response developed thereby play a crucial role in developing resistance against <i>C. neoformans</i>; (b) Pep1p, an aspartic protease as well as an antigenic protein secreted by <i>C. neoformans</i>, can be exploited for vaccination (both prophylactic and therapeutic) or immunotherapy to improve the host defense during this fungal infection.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142503520","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}
Claire M Doherty, Paige R Patterson, Julie A Emeanuwa, Jessica Belmares Ortega, Barbara A Fox, David J Bzik, Eric Y Denkers
<p><p>As a model organism in the study of immunity to infection, <i>Toxoplasma gondii</i> has been instrumental in establishing key principles of host anti-microbial defense and its regulation. Here, we employed an attenuated uracil auxotroph strain of Type I <i>Toxoplasma</i> designated OMP to further untangle the early immune response to this parasitic pathogen. Experiments using αβ T cell-deficient <i>Tcrb</i><sup><i>-/-</i></sup> mice unexpectedly revealed that an intact αβ T lymphocyte compartment was essential to survive infection with OMP. Subsequent antibody depletion and knockout mouse experiments demonstrated contributions from CD4<sup>+</sup> T cells and most predominantly CD8<sup>+</sup> T cells in resistance. Using transgenic knockout mice, we found only a partial requirement for IFN-γ and a lack of requirement for Toll-like receptor (TLR) adaptor MyD88 in resistance. In contrast to other studies on <i>Toxoplasma</i>, the ability to survive OMP infection did not require IL-12p40. Surprisingly, T cell-dependent IL-10 was found to be critical for survival, and deficiency of this cytokine triggered an abnormally high systemic inflammatory response. We also found that parasite molecule GRA24, a dense granule protein that triggers TLR-independent IL-12 production, acts as a virulence factor contributing to death of OMP-infected <i>Tcrb</i><sup><i>-/-</i></sup> and <i>IL-10</i><sup><i>-/-</i></sup> mice. Furthermore, resistance against OMP was restored in <i>Tcrb</i><sup><i>-/-</i></sup> mice via monoclonal depletion of IL-12p40, suggesting that GRA24-induced IL-12 underlies the fatal immunopathology observed. Collectively, our studies provide insight into a novel and rapidly arising T lymphocyte-dependent anti-inflammatory response to <i>T. gondii</i> which operates independently of MyD88 and IL-12 and that depends on the function of parasite-dense granule protein GRA24.IMPORTANCEAs a model infectious microbe and an important human pathogen, the apicomplexan <i>Toxoplasma gondii</i> has provided many important insights into innate and adaptive immunity to infection. We show here that a low virulence uracil auxotrophic <i>Toxoplasma</i> strain emerges as a virulent parasite in the absence of an intact T cell compartment. Both CD4<sup>+</sup> and CD8<sup>+</sup> T lymphocytes are required for optimal protection, in line with previous findings in other models of <i>Toxoplasma</i> infection. Nevertheless, several novel aspects of the response were identified in our study. Protection occurs independently of IL-12 and MyD88 and only partially requires IFN-γ. This is noteworthy particularly because the cytokines IL-12 and IFN-γ have previously been regarded as essential for protective immunity to <i>T. gondii</i>. Instead, we identified the anti-inflammatory effects of T cell-dependent IL-10 as the critical factor enabling host survival. The parasite dense granule protein GRA24, a host-directed mitogen-activated protein kinase activator, was ide
作为研究感染免疫的模式生物,弓形虫在建立宿主抗微生物防御及其调控的关键原则方面发挥了重要作用。在这里,我们采用了被命名为 OMP 的 I 型弓形虫尿嘧啶辅助减毒株,以进一步解开这种寄生病原体的早期免疫反应。使用αβ T细胞缺陷的Tcrb-/-小鼠进行的实验意外地发现,完整的αβ T淋巴细胞区系是在感染OMP后存活的必要条件。随后的抗体耗竭和基因敲除小鼠实验表明,CD4+ T 细胞和最主要的 CD8+ T 细胞对抵抗力有贡献。通过使用转基因基因敲除小鼠,我们发现在抗药性中只需要部分 IFN-γ,而不需要 Toll 样受体(TLR)适配体 MyD88。与其他有关弓形虫的研究不同,OMP 感染的存活能力并不需要 IL-12p40。令人惊讶的是,我们发现依赖于 T 细胞的 IL-10 对存活至关重要,缺乏这种细胞因子会引发异常高的全身炎症反应。我们还发现,寄生虫分子 GRA24 是一种致密颗粒蛋白,它能触发不依赖于 TLR 的 IL-12 的产生,是导致感染了 OMP 的 Tcrb-/- 和 IL-10-/- 小鼠死亡的毒力因子。此外,Tcrb-/-小鼠通过单克隆去除IL-12p40可恢复对OMP的抵抗力,这表明GRA24诱导的IL-12是观察到的致命免疫病理学的基础。总之,我们的研究深入揭示了一种新的、快速产生的依赖于 T 淋巴细胞的对弓形虫的抗炎反应,这种反应独立于 MyD88 和 IL-12 而运行,并且依赖于寄生虫致密颗粒蛋白 GRA24 的功能。重要意义弓形虫作为一种模式传染性微生物和重要的人类病原体,为先天性和适应性免疫感染提供了许多重要的见解。我们在此表明,在没有完整的 T 细胞区系的情况下,低毒性尿嘧啶辅助营养性弓形虫菌株会成为一种毒性寄生虫。CD4+ 和 CD8+ T 淋巴细胞都是最佳保护所必需的,这与之前在其他弓形虫感染模型中的发现一致。然而,我们的研究发现了反应的几个新方面。保护作用的产生与 IL-12 和 MyD88 无关,仅部分需要 IFN-γ。这一点尤其值得注意,因为细胞因子IL-12和IFN-γ以前一直被认为是对淋球菌产生保护性免疫的必要条件。相反,我们发现依赖于 T 细胞的 IL-10 的抗炎作用是使宿主存活的关键因素。寄生虫致密颗粒蛋白 GRA24 是宿主定向的丝裂原活化蛋白激酶激活剂,被确定为 T 细胞缺陷宿主的主要毒力因子。总之,我们的研究结果为了解宿主对弓形虫的抵抗力提供了意想不到的新见解。
{"title":"T lymphocyte-dependent IL-10 down-regulates a cytokine storm driven by <i>Toxoplasma gondii</i> GRA24.","authors":"Claire M Doherty, Paige R Patterson, Julie A Emeanuwa, Jessica Belmares Ortega, Barbara A Fox, David J Bzik, Eric Y Denkers","doi":"10.1128/mbio.01455-24","DOIUrl":"https://doi.org/10.1128/mbio.01455-24","url":null,"abstract":"<p><p>As a model organism in the study of immunity to infection, <i>Toxoplasma gondii</i> has been instrumental in establishing key principles of host anti-microbial defense and its regulation. Here, we employed an attenuated uracil auxotroph strain of Type I <i>Toxoplasma</i> designated OMP to further untangle the early immune response to this parasitic pathogen. Experiments using αβ T cell-deficient <i>Tcrb</i><sup><i>-/-</i></sup> mice unexpectedly revealed that an intact αβ T lymphocyte compartment was essential to survive infection with OMP. Subsequent antibody depletion and knockout mouse experiments demonstrated contributions from CD4<sup>+</sup> T cells and most predominantly CD8<sup>+</sup> T cells in resistance. Using transgenic knockout mice, we found only a partial requirement for IFN-γ and a lack of requirement for Toll-like receptor (TLR) adaptor MyD88 in resistance. In contrast to other studies on <i>Toxoplasma</i>, the ability to survive OMP infection did not require IL-12p40. Surprisingly, T cell-dependent IL-10 was found to be critical for survival, and deficiency of this cytokine triggered an abnormally high systemic inflammatory response. We also found that parasite molecule GRA24, a dense granule protein that triggers TLR-independent IL-12 production, acts as a virulence factor contributing to death of OMP-infected <i>Tcrb</i><sup><i>-/-</i></sup> and <i>IL-10</i><sup><i>-/-</i></sup> mice. Furthermore, resistance against OMP was restored in <i>Tcrb</i><sup><i>-/-</i></sup> mice via monoclonal depletion of IL-12p40, suggesting that GRA24-induced IL-12 underlies the fatal immunopathology observed. Collectively, our studies provide insight into a novel and rapidly arising T lymphocyte-dependent anti-inflammatory response to <i>T. gondii</i> which operates independently of MyD88 and IL-12 and that depends on the function of parasite-dense granule protein GRA24.IMPORTANCEAs a model infectious microbe and an important human pathogen, the apicomplexan <i>Toxoplasma gondii</i> has provided many important insights into innate and adaptive immunity to infection. We show here that a low virulence uracil auxotrophic <i>Toxoplasma</i> strain emerges as a virulent parasite in the absence of an intact T cell compartment. Both CD4<sup>+</sup> and CD8<sup>+</sup> T lymphocytes are required for optimal protection, in line with previous findings in other models of <i>Toxoplasma</i> infection. Nevertheless, several novel aspects of the response were identified in our study. Protection occurs independently of IL-12 and MyD88 and only partially requires IFN-γ. This is noteworthy particularly because the cytokines IL-12 and IFN-γ have previously been regarded as essential for protective immunity to <i>T. gondii</i>. Instead, we identified the anti-inflammatory effects of T cell-dependent IL-10 as the critical factor enabling host survival. The parasite dense granule protein GRA24, a host-directed mitogen-activated protein kinase activator, was ide","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142503528","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}
Benjamin S Steadman, Jifeng Bian, Ronald A Shikiya, Jason C Bartz
Mammalian prion diseases are infectious neurodegenerative diseases caused by the self-templating form of the prion protein PrPSc. Much evidence supports the hypothesis that prions exist as a mixture of a dominant strain and minor prion strains. While it is known that prions can infect new species, the relative contribution of the dominant prion strain and minor strains in crossing the species barrier is unknown. We previously identified minor prion strains from a biologically cloned drowsy (DY) strain of hamster-adapted transmissible mink encephalopathy (TME). Here we show that these minor prion strains have increased infection efficiency to rabbit kidney epithelial cells that express hamster PrPC compared to the dominant strain DY TME. Using protein misfolding cyclic amplification (PMCA), we found that the dominant strain DY TME failed to convert mouse PrPC to PrPSc, even after several serial passages. In contrast, the minor prion strains isolated from biologically cloned DY TME robustly converted mouse PrPC to PrPSc in the first round of PMCA. This observation indicates that minor prion strains from the mutant spectra contribute to crossing the species barrier. Additionally, we found that the PMCA conversion efficiency for the minor prion strains tested was significantly different from each other and from the short-incubation period prion strain HY TME. This suggests that minor strain diversity may be greater than previously anticipated. These observations further expand our understanding of the mechanisms underlying the species barrier effect and has implications for assessing the zoonotic potential of prions.
Importance: Prions from cattle with bovine spongiform encephalopathy have transmitted to humans, whereas scrapie from sheep and goats likely has not, suggesting that some prions can cross species barriers more easily than others. Prions are composed of a dominant strain and minor strains, and the contribution of each population to adapt to new replicative environments is unknown. Recently, minor prion strains were isolated from the biologically cloned prion strain DY TME, and these minor prion strains differed in properties from the dominant prion strain, DY TME. Here we found that these minor prion strains also differed in conversion efficiency and host range compared to the dominant strain DY TME. These novel findings provide evidence that minor prion strains contribute to interspecies transmission, underscoring the significance of minor strain components in important biological processes.
哺乳动物朊病毒病是由朊病毒蛋白 PrPSc 的自模板形式引起的传染性神经退行性疾病。许多证据都支持朊病毒以显性菌株和次要朊病毒菌株混合物的形式存在的假说。虽然人们知道朊病毒可以感染新物种,但显性朊病毒菌株和次要菌株在跨越物种屏障方面的相对贡献尚不清楚。我们先前从仓鼠适应性传染性水貂脑病(TME)的生物克隆昏睡(DY)株中鉴定出了次要朊病毒株。在这里,我们发现与优势菌株 DY TME 相比,这些次要朊病毒菌株对表达仓鼠 PrPC 的兔肾上皮细胞的感染效率更高。通过使用蛋白质错折叠循环扩增(PMCA),我们发现显性菌株 DY TME 即使经过多次连续传代,也无法将小鼠 PrPC 转化为 PrPSc。与此相反,从生物克隆的 DY TME 中分离出的次要朊病毒菌株在第一轮 PMCA 中就能将小鼠 PrPC 强健地转化为 PrPSc。这一观察结果表明,来自突变体谱系的小朊病毒菌株有助于跨越物种屏障。此外,我们还发现,所测试的次要朊病毒菌株的 PMCA 转化效率与短孵育期朊病毒菌株 HY TME 之间存在显著差异。这表明次要菌株的多样性可能比先前预期的要大。这些观察结果进一步拓展了我们对物种屏障效应机制的理解,并对评估朊病毒的人畜共患可能性具有重要意义:重要意义:牛海绵状脑病患者的朊病毒曾传播给人类,而绵羊和山羊的瘙痒病可能没有传播给人类,这表明某些朊病毒比其他朊病毒更容易跨越物种屏障。朊病毒由优势菌株和次优势菌株组成,每个种群在适应新复制环境方面的贡献尚不清楚。最近,从生物克隆的朊病毒菌株 DY TME 中分离出了次要朊病毒菌株,这些次要朊病毒菌株的特性与优势朊病毒菌株 DY TME 不同。我们在这里发现,与优势菌株 DY TME 相比,这些次要朊病毒菌株在转化效率和宿主范围方面也有所不同。这些新发现提供了次要朊病毒菌株有助于物种间传播的证据,强调了次要菌株成分在重要生物过程中的重要性。
{"title":"Minor prion substrains overcome transmission barriers.","authors":"Benjamin S Steadman, Jifeng Bian, Ronald A Shikiya, Jason C Bartz","doi":"10.1128/mbio.02721-24","DOIUrl":"https://doi.org/10.1128/mbio.02721-24","url":null,"abstract":"<p><p>Mammalian prion diseases are infectious neurodegenerative diseases caused by the self-templating form of the prion protein PrP<sup>Sc</sup>. Much evidence supports the hypothesis that prions exist as a mixture of a dominant strain and minor prion strains. While it is known that prions can infect new species, the relative contribution of the dominant prion strain and minor strains in crossing the species barrier is unknown. We previously identified minor prion strains from a biologically cloned drowsy (DY) strain of hamster-adapted transmissible mink encephalopathy (TME). Here we show that these minor prion strains have increased infection efficiency to rabbit kidney epithelial cells that express hamster PrP<sup>C</sup> compared to the dominant strain DY TME. Using protein misfolding cyclic amplification (PMCA), we found that the dominant strain DY TME failed to convert mouse PrP<sup>C</sup> to PrP<sup>Sc</sup>, even after several serial passages. In contrast, the minor prion strains isolated from biologically cloned DY TME robustly converted mouse PrP<sup>C</sup> to PrP<sup>Sc</sup> in the first round of PMCA. This observation indicates that minor prion strains from the mutant spectra contribute to crossing the species barrier. Additionally, we found that the PMCA conversion efficiency for the minor prion strains tested was significantly different from each other and from the short-incubation period prion strain HY TME. This suggests that minor strain diversity may be greater than previously anticipated. These observations further expand our understanding of the mechanisms underlying the species barrier effect and has implications for assessing the zoonotic potential of prions.</p><p><strong>Importance: </strong>Prions from cattle with bovine spongiform encephalopathy have transmitted to humans, whereas scrapie from sheep and goats likely has not, suggesting that some prions can cross species barriers more easily than others. Prions are composed of a dominant strain and minor strains, and the contribution of each population to adapt to new replicative environments is unknown. Recently, minor prion strains were isolated from the biologically cloned prion strain DY TME, and these minor prion strains differed in properties from the dominant prion strain, DY TME. Here we found that these minor prion strains also differed in conversion efficiency and host range compared to the dominant strain DY TME. These novel findings provide evidence that minor prion strains contribute to interspecies transmission, underscoring the significance of minor strain components in important biological processes.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142503527","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}
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":"https://doi.org/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":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jeffrey M Boyd, Kylie Ryan Kaler, Karla Esquilín-Lebrón, Ashley Pall, Courtney J Campbell, Mary E Foley, Gustavo Rios-Delgado, Emilee M Mustor, Timothy G Stephens, Hannah Bovermann, Todd M Greco, Ileana M Cristea, Valerie J Carabetta, William N Beavers, Debashish Bhattacharya, Eric P Skaar, Lindsey N Shaw, Timothy L Stemmler
Iron (Fe) is a trace nutrient required by nearly all organisms. As a result of the demand for Fe and the toxicity of non-chelated cytosolic ionic Fe, regulatory systems have evolved to tightly balance Fe acquisition and usage while limiting overload. In most bacteria, including the mammalian pathogen Staphylococcus aureus, the ferric uptake regulator (Fur) is the primary transcriptional regulator controlling the transcription of genes that code for Fe uptake and utilization proteins. Fpa (formerly YlaN) was demonstrated to be essential in Bacillus subtilis unless excess Fe is added to the growth medium, suggesting a role in Fe homeostasis. Here, we demonstrate that Fpa is essential in S. aureus upon Fe deprivation. Null fur alleles bypassed the essentiality of Fpa. The absence of Fpa abolished the derepression of Fur-regulated genes during Fe limitation. Bioinformatic analyses suggest that fpa was recruited to Gram-positive bacteria and, once acquired, was maintained in the genome as it co-evolved with Fur. Consistent with a role for Fpa in alleviating Fur-dependent repression, Fpa and Fur interacted in vivo, and Fpa decreased the DNA-binding ability of Fur in vitro. Fpa bound Fe(II) in vitro using oxygen or nitrogen ligands with an association constant that is consistent with a physiological role in Fe homeostasis. These findings have led to a model wherein Fpa is an Fe(II) binding protein that influences Fur-dependent regulation through direct interaction.IMPORTANCEIron (Fe) is an essential nutrient for nearly all organisms. If Fe homeostasis is not maintained, Fe may accumulate in the cytosol, which can be toxic. Questions remain about how cells efficiently balance Fe uptake and usage to prevent overload. Iron uptake and proper metalation of proteins are essential processes in the mammalian bacterial pathogen Staphylococcus aureus. Understanding the gene products involved in the genetic regulation of Fe uptake and usage and the physiological adaptations that S. aureus uses to survive in Fe-depleted conditions provides insight into pathogenesis. Herein, we demonstrate that the DNA-binding activity of the ferric uptake regulator transcriptional repressor is alleviated under Fe limitation, but uniquely, in S. aureus, alleviation requires the presence of Fpa.
{"title":"Fpa (YlaN) is an iron(II) binding protein that functions to relieve Fur-mediated repression of gene expression in <i>Staphylococcus aureus</i>.","authors":"Jeffrey M Boyd, Kylie Ryan Kaler, Karla Esquilín-Lebrón, Ashley Pall, Courtney J Campbell, Mary E Foley, Gustavo Rios-Delgado, Emilee M Mustor, Timothy G Stephens, Hannah Bovermann, Todd M Greco, Ileana M Cristea, Valerie J Carabetta, William N Beavers, Debashish Bhattacharya, Eric P Skaar, Lindsey N Shaw, Timothy L Stemmler","doi":"10.1128/mbio.02310-24","DOIUrl":"https://doi.org/10.1128/mbio.02310-24","url":null,"abstract":"<p><p>Iron (Fe) is a trace nutrient required by nearly all organisms. As a result of the demand for Fe and the toxicity of non-chelated cytosolic ionic Fe, regulatory systems have evolved to tightly balance Fe acquisition and usage while limiting overload. In most bacteria, including the mammalian pathogen <i>Staphylococcus aureus</i>, the ferric uptake regulator (Fur) is the primary transcriptional regulator controlling the transcription of genes that code for Fe uptake and utilization proteins. Fpa (formerly YlaN) was demonstrated to be essential in <i>Bacillus subtilis</i> unless excess Fe is added to the growth medium, suggesting a role in Fe homeostasis. Here, we demonstrate that Fpa is essential in <i>S. aureus</i> upon Fe deprivation. Null <i>fur</i> alleles bypassed the essentiality of Fpa. The absence of Fpa abolished the derepression of Fur-regulated genes during Fe limitation. Bioinformatic analyses suggest that <i>fpa</i> was recruited to Gram-positive bacteria and, once acquired, was maintained in the genome as it co-evolved with Fur. Consistent with a role for Fpa in alleviating Fur-dependent repression, Fpa and Fur interacted <i>in vivo</i>, and Fpa decreased the DNA-binding ability of Fur <i>in vitro</i>. Fpa bound Fe(II) <i>in vitro</i> using oxygen or nitrogen ligands with an association constant that is consistent with a physiological role in Fe homeostasis. These findings have led to a model wherein Fpa is an Fe(II) binding protein that influences Fur-dependent regulation through direct interaction.IMPORTANCEIron (Fe) is an essential nutrient for nearly all organisms. If Fe homeostasis is not maintained, Fe may accumulate in the cytosol, which can be toxic. Questions remain about how cells efficiently balance Fe uptake and usage to prevent overload. Iron uptake and proper metalation of proteins are essential processes in the mammalian bacterial pathogen <i>Staphylococcus aureus</i>. Understanding the gene products involved in the genetic regulation of Fe uptake and usage and the physiological adaptations that <i>S. aureus</i> uses to survive in Fe-depleted conditions provides insight into pathogenesis. Herein, we demonstrate that the DNA-binding activity of the ferric uptake regulator transcriptional repressor is alleviated under Fe limitation, but uniquely, in <i>S. aureus</i>, alleviation requires the presence of Fpa.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142503524","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}
Erika R Johansen, Damon L Schmalzriedt, Danilela Avila, Paul A Sylvester, Cade R Rahlf, Jordan M Bobek, Daisy Sahoo, Bonnie N Dittel, Vera L Tarakanova
Gammaherpesviruses are species-specific, ubiquitous pathogens that establish lifelong infection in their hosts and are associated with cancers, including B cell lymphomas. Type I and II interferons (IFNs) are critical for the control of acute and chronic gammaherpesvirus infection. However, the cell type-specific role of IFN signaling during natural infection is poorly defined and is masked by the altered viral pathogenesis observed in hosts with global IFN deficiencies. STAT1 is a constitutively expressed transcription factor that is critical for the effector function of type I and II IFNs. In this study, we defined the impact of B cell-specific STAT1 expression on gammaherpesvirus infection using murine gammaherpesvirus 68 (MHV68). While the acute stage of MHV68 infection was not affected, we found opposite, anatomic site-dependent effects of B cell-intrinsic STAT1 expression during chronic infection. Consistent with the antiviral role of STAT1, B cell-specific STAT1 expression attenuated the latent viral reservoir in peritoneal B cells of chronically infected mice. In contrast, STAT1 expression in splenic B cells supported the establishment of the latent MHV68 reservoir in germinal center B cells, revealing an unexpected proviral role of B cell-intrinsic STAT1 expression during chronic gammaherpesvirus infection. These STAT1-dependent MHV68 chronic infection phenotypes were fully recapitulated in the peritoneal cavity but not the spleen of mice with B cell-specific deficiency of type I IFN receptor. In summary, our study uncovers the intriguing combination of proviral and antiviral roles of B cell-intrinsic STAT1 expression during chronic gammaherpesvirus infection.IMPORTANCEInterferons (IFNs) execute broadly antiviral roles during acute and chronic viral infections. The constitutively expressed transcription factor STAT1 is a critical downstream effector of IFN signaling. Our studies demonstrate that B cell-intrinsic STAT1 expression has opposing and anatomic site-dependent roles during chronic gammaherpesvirus infection. Specifically, B cell-intrinsic STAT1 expression restricted gammaherpesvirus latent reservoir in the peritoneal cavity, consistent with the classical antiviral role of STAT1. In contrast, decreased STAT1 expression in splenic B cells led to the attenuated establishment of gammaherpesvirus latency and decreased latent infection of germinal center B cells, highlighting a novel proviral role of B cell-intrinsic STAT1 expression during chronic infection with a B cell-tropic gammaherpesvirus. Interestingly, B cell-specific type I IFN receptor deficiency primarily recapitulated the antiviral role of B cell-intrinsic STAT1 expression, suggesting the compensatory function of B cell-intrinsic type II IFN signaling or an IFN-independent proviral role of B cell-intrinsic STAT1 expression during chronic gammaherpesvirus infection.
γ疱疹病毒是物种特异性的、无处不在的病原体,可在宿主体内建立终生感染,并与癌症(包括 B 细胞淋巴瘤)有关。I 型和 II 型干扰素(IFNs)对控制急性和慢性γ疱疹病毒感染至关重要。然而,在自然感染过程中,IFN 信号传导的细胞类型特异性作用尚不明确,而且在全面缺乏 IFN 的宿主中观察到的病毒发病机制的改变也掩盖了这一作用。STAT1 是一种组成型表达的转录因子,对 I 型和 II 型 IFN 的效应功能至关重要。在本研究中,我们利用鼠γ疱疹病毒 68(MHV68)确定了 B 细胞特异性 STAT1 表达对γ疱疹病毒感染的影响。虽然 MHV68 感染的急性期不受影响,但我们发现在慢性感染期间,B 细胞内在 STAT1 的表达会产生相反的、解剖部位依赖性的影响。与 STAT1 的抗病毒作用相一致,B 细胞特异性 STAT1 的表达削弱了慢性感染小鼠腹膜 B 细胞中的潜伏病毒库。与此相反,脾脏 B 细胞中 STAT1 的表达支持了生殖中心 B 细胞中潜伏 MHV68 病毒库的建立,揭示了 B 细胞内在 STAT1 表达在慢性γ疱疹病毒感染过程中起到了意想不到的抑制病毒作用。这些 STAT1 依赖性 MHV68 慢性感染表型在 B 细胞特异性 IFN 受体缺乏的小鼠腹腔而非脾脏中完全重现。重要意义干扰素(IFNs)在急性和慢性病毒感染期间发挥着广泛的抗病毒作用。组成型表达的转录因子 STAT1 是 IFN 信号转导的关键下游效应器。我们的研究表明,在慢性γ疱疹病毒感染期间,B细胞内STAT1的表达具有对立和解剖部位依赖性的作用。具体来说,B 细胞内在 STAT1 表达限制了腹腔中的γ疱疹病毒潜伏库,这与 STAT1 的经典抗病毒作用一致。与此相反,脾脏 B 细胞中 STAT1 表达的减少导致了伽马疱疹病毒潜伏期的建立和生殖中心 B 细胞潜伏感染的减少,突出了 B 细胞内在 STAT1 表达在 B 细胞致病性伽马疱疹病毒慢性感染过程中的新的病毒作用。有趣的是,B细胞特异性I型IFN受体缺乏主要重现了B细胞内在STAT1表达的抗病毒作用,这表明在慢性γ疱疹病毒感染期间,B细胞内在II型IFN信号传导具有补偿功能,或B细胞内在STAT1表达具有独立于IFN的挑衅性作用。
{"title":"Combination of proviral and antiviral roles of B cell-intrinsic STAT1 expression defines parameters of chronic gammaherpesvirus infection.","authors":"Erika R Johansen, Damon L Schmalzriedt, Danilela Avila, Paul A Sylvester, Cade R Rahlf, Jordan M Bobek, Daisy Sahoo, Bonnie N Dittel, Vera L Tarakanova","doi":"10.1128/mbio.01598-24","DOIUrl":"https://doi.org/10.1128/mbio.01598-24","url":null,"abstract":"<p><p>Gammaherpesviruses are species-specific, ubiquitous pathogens that establish lifelong infection in their hosts and are associated with cancers, including B cell lymphomas. Type I and II interferons (IFNs) are critical for the control of acute and chronic gammaherpesvirus infection. However, the cell type-specific role of IFN signaling during natural infection is poorly defined and is masked by the altered viral pathogenesis observed in hosts with global IFN deficiencies. STAT1 is a constitutively expressed transcription factor that is critical for the effector function of type I and II IFNs. In this study, we defined the impact of B cell-specific STAT1 expression on gammaherpesvirus infection using murine gammaherpesvirus 68 (MHV68). While the acute stage of MHV68 infection was not affected, we found opposite, anatomic site-dependent effects of B cell-intrinsic STAT1 expression during chronic infection. Consistent with the antiviral role of STAT1, B cell-specific STAT1 expression attenuated the latent viral reservoir in peritoneal B cells of chronically infected mice. In contrast, STAT1 expression in splenic B cells supported the establishment of the latent MHV68 reservoir in germinal center B cells, revealing an unexpected proviral role of B cell-intrinsic STAT1 expression during chronic gammaherpesvirus infection. These STAT1-dependent MHV68 chronic infection phenotypes were fully recapitulated in the peritoneal cavity but not the spleen of mice with B cell-specific deficiency of type I IFN receptor. In summary, our study uncovers the intriguing combination of proviral and antiviral roles of B cell-intrinsic STAT1 expression during chronic gammaherpesvirus infection.IMPORTANCEInterferons (IFNs) execute broadly antiviral roles during acute and chronic viral infections. The constitutively expressed transcription factor STAT1 is a critical downstream effector of IFN signaling. Our studies demonstrate that B cell-intrinsic STAT1 expression has opposing and anatomic site-dependent roles during chronic gammaherpesvirus infection. Specifically, B cell-intrinsic STAT1 expression restricted gammaherpesvirus latent reservoir in the peritoneal cavity, consistent with the classical antiviral role of STAT1. In contrast, decreased STAT1 expression in splenic B cells led to the attenuated establishment of gammaherpesvirus latency and decreased latent infection of germinal center B cells, highlighting a novel proviral role of B cell-intrinsic STAT1 expression during chronic infection with a B cell-tropic gammaherpesvirus. Interestingly, B cell-specific type I IFN receptor deficiency primarily recapitulated the antiviral role of B cell-intrinsic STAT1 expression, suggesting the compensatory function of B cell-intrinsic type II IFN signaling or an IFN-independent proviral role of B cell-intrinsic STAT1 expression during chronic gammaherpesvirus infection.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142503522","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}
Jacob Burch-Konda, Biraj B Kayastha, Myriam Achour, Aya Kubo, Mackenzie Hull, Reygan Braga, Lorelei Winton, Rendi R Rogers, Erika I Lutter, Marianna A Patrauchan
<p><p>The human pathogen <i>Pseudomonas aeruginosa</i> (<i>Pa</i>) poses a major risk for a range of severe infections, particularly lung infections in patients suffering from cystic fibrosis (CF). As previously reported, the virulent behavior of this pathogen is enhanced by elevated levels of Ca<sup>2+</sup> that are commonly present in CF nasal and lung fluids. In addition, a Ca<sup>2+</sup>-binding EF-hand protein, EfhP (PA4107), was partially characterized and shown to be critical for the Ca<sup>2+</sup>-regulated virulence in <i>P. aeruginosa</i>. Here, we describe the rapid (10 min, 60 min), and adaptive (12 h) transcriptional responses of PAO1 to elevated Ca<sup>2+</sup> detected by genome-wide RNA sequencing and show that <i>efhP</i> deletion significantly hindered both rapid and adaptive Ca<sup>2+</sup> regulation. The most differentially regulated genes included multiple Fe sequestering mechanisms, a large number of extracytoplasmic function sigma factors (ECFσ), and several virulence factors, such as the production of pyocins. The Ca<sup>2+</sup> regulation of Fe uptake was also observed in CF clinical isolates and appeared to involve the global regulator Fur. In addition, we showed that the <i>efhP</i> transcription is controlled by Ca<sup>2+</sup> and Fe, and this regulation required a Ca<sup>2+</sup>-dependent two-component regulatory system CarSR. Furthermore, the <i>efhP</i> expression is significantly increased in CF clinical isolates and upon pathogen internalization into epithelial cells. Overall, the results established for the first time that Ca<sup>2+</sup> controls Fe sequestering mechanisms in <i>P. aeruginosa</i> and that EfhP plays a key role in the regulatory interconnectedness between Ca<sup>2+</sup> and Fe signaling pathways, the two distinct and important signaling pathways that guide the pathogen's adaptation to the host.IMPORTANCE<i>Pseudomonas aeruginosa</i> (<i>Pa</i>) poses a major risk for severe infections, particularly in patients suffering from cystic fibrosis (CF). For the first time, kinetic RNA sequencing analysis identified <i>Pa</i> rapid and adaptive transcriptional responses to Ca<sup>2+</sup> levels consistent with those present in CF respiratory fluids. The most highly upregulated processes include iron sequestering, iron starvation sigma factors, and self-lysis factors pyocins. An EF-hand Ca<sup>2+</sup> sensor, EfhP, is required for at least 1/3 of the Ca<sup>2+</sup> response, including the majority of the iron uptake mechanisms and the production of pyocins. Transcription of <i>efhP</i> itself is regulated by Ca<sup>2+</sup> and Fe, and increases during interactions with host epithelial cells, suggesting the protein's important role in <i>Pa</i> infections. The findings establish the regulatory interconnectedness between Ca<sup>2+</sup> and iron signaling pathways that shape <i>Pa</i> transcriptional responses. Therefore, understanding Pa's transcriptional response to Ca<sup>2+</sup> and associa
{"title":"EF-hand calcium sensor, EfhP, controls transcriptional regulation of iron uptake by calcium in <i>Pseudomonas aeruginosa</i>.","authors":"Jacob Burch-Konda, Biraj B Kayastha, Myriam Achour, Aya Kubo, Mackenzie Hull, Reygan Braga, Lorelei Winton, Rendi R Rogers, Erika I Lutter, Marianna A Patrauchan","doi":"10.1128/mbio.02447-24","DOIUrl":"10.1128/mbio.02447-24","url":null,"abstract":"<p><p>The human pathogen <i>Pseudomonas aeruginosa</i> (<i>Pa</i>) poses a major risk for a range of severe infections, particularly lung infections in patients suffering from cystic fibrosis (CF). As previously reported, the virulent behavior of this pathogen is enhanced by elevated levels of Ca<sup>2+</sup> that are commonly present in CF nasal and lung fluids. In addition, a Ca<sup>2+</sup>-binding EF-hand protein, EfhP (PA4107), was partially characterized and shown to be critical for the Ca<sup>2+</sup>-regulated virulence in <i>P. aeruginosa</i>. Here, we describe the rapid (10 min, 60 min), and adaptive (12 h) transcriptional responses of PAO1 to elevated Ca<sup>2+</sup> detected by genome-wide RNA sequencing and show that <i>efhP</i> deletion significantly hindered both rapid and adaptive Ca<sup>2+</sup> regulation. The most differentially regulated genes included multiple Fe sequestering mechanisms, a large number of extracytoplasmic function sigma factors (ECFσ), and several virulence factors, such as the production of pyocins. The Ca<sup>2+</sup> regulation of Fe uptake was also observed in CF clinical isolates and appeared to involve the global regulator Fur. In addition, we showed that the <i>efhP</i> transcription is controlled by Ca<sup>2+</sup> and Fe, and this regulation required a Ca<sup>2+</sup>-dependent two-component regulatory system CarSR. Furthermore, the <i>efhP</i> expression is significantly increased in CF clinical isolates and upon pathogen internalization into epithelial cells. Overall, the results established for the first time that Ca<sup>2+</sup> controls Fe sequestering mechanisms in <i>P. aeruginosa</i> and that EfhP plays a key role in the regulatory interconnectedness between Ca<sup>2+</sup> and Fe signaling pathways, the two distinct and important signaling pathways that guide the pathogen's adaptation to the host.IMPORTANCE<i>Pseudomonas aeruginosa</i> (<i>Pa</i>) poses a major risk for severe infections, particularly in patients suffering from cystic fibrosis (CF). For the first time, kinetic RNA sequencing analysis identified <i>Pa</i> rapid and adaptive transcriptional responses to Ca<sup>2+</sup> levels consistent with those present in CF respiratory fluids. The most highly upregulated processes include iron sequestering, iron starvation sigma factors, and self-lysis factors pyocins. An EF-hand Ca<sup>2+</sup> sensor, EfhP, is required for at least 1/3 of the Ca<sup>2+</sup> response, including the majority of the iron uptake mechanisms and the production of pyocins. Transcription of <i>efhP</i> itself is regulated by Ca<sup>2+</sup> and Fe, and increases during interactions with host epithelial cells, suggesting the protein's important role in <i>Pa</i> infections. The findings establish the regulatory interconnectedness between Ca<sup>2+</sup> and iron signaling pathways that shape <i>Pa</i> transcriptional responses. Therefore, understanding Pa's transcriptional response to Ca<sup>2+</sup> and associa","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142469437","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}
Jeongjoon Choi, Kaitlyn Speckhart, Billy Tsai, Daniel DiMaio
Rab GTPases control intracellular vesicular transport, including retrograde trafficking of human papillomavirus (HPV) during cell entry, guiding the virus from the endosome to the trans-Golgi network (TGN), the Golgi apparatus, and eventually the nucleus. Rab proteins have been identified that act prior to the arrival of HPV at the TGN, but Rab proteins operating in later stages of entry remain elusive. Here, we report that knockdown of Rab6a impairs HPV entry by preventing HPV exit from the TGN and impeding intra-Golgi transport of the incoming virus. Rab6a supports HPV trafficking by facilitating the association of HPV with dynein, a motor protein complex, and BICD2, a dynein adaptor, in the TGN. L2 can bind directly to GTP-Rab6a in vitro, and excess of either GTP-Rab6a or GDP-Rab6 inhibits HPV entry, suggesting that cycling between GDP-Rab6 and GTP-Rab6 is critical. Notably, Rab6a is crucial for HPV-BICD2 and HPV-dynein association in the TGN of infected cells but not in the endosome. Our findings reveal important features of the molecular basis of HPV infection, including the discovery that HPV uses different mechanisms to engage dynein at different times during entry, and identify potential targets for therapeutic approaches to inhibit HPV infection.
Importance: Human papillomaviruses (HPVs) are small, non-enveloped DNA viruses that cause approximately 5% of human cancer. Like most other DNA viruses, HPV traffics to the nucleus during virus entry to successfully infect cells. We show here that HPV utilizes a cellular enzyme, Rab6a, during virus entry to engage the dynein molecular motor for transport along microtubules. Rab6a is required for complex formation between the HPV L2 capsid protein, dynein, and the dynein adaptor BICD2 in the trans-Golgi network (TGN). This complex is required for transport of the incoming virus out of the TGN as it journeys to the nucleus. Our findings identify potential targets for therapeutic approaches.
{"title":"Rab6a enables BICD2/dynein-mediated trafficking of human papillomavirus from the <i>trans-</i>Golgi network during virus entry.","authors":"Jeongjoon Choi, Kaitlyn Speckhart, Billy Tsai, Daniel DiMaio","doi":"10.1128/mbio.02811-24","DOIUrl":"10.1128/mbio.02811-24","url":null,"abstract":"<p><p>Rab GTPases control intracellular vesicular transport, including retrograde trafficking of human papillomavirus (HPV) during cell entry, guiding the virus from the endosome to the <i>trans-</i>Golgi network (TGN), the Golgi apparatus, and eventually the nucleus. Rab proteins have been identified that act prior to the arrival of HPV at the TGN, but Rab proteins operating in later stages of entry remain elusive. Here, we report that knockdown of Rab6a impairs HPV entry by preventing HPV exit from the TGN and impeding intra-Golgi transport of the incoming virus. Rab6a supports HPV trafficking by facilitating the association of HPV with dynein, a motor protein complex, and BICD2, a dynein adaptor, in the TGN. L2 can bind directly to GTP-Rab6a <i>in vitro</i>, and excess of either GTP-Rab6a or GDP-Rab6 inhibits HPV entry, suggesting that cycling between GDP-Rab6 and GTP-Rab6 is critical. Notably, Rab6a is crucial for HPV-BICD2 and HPV-dynein association in the TGN of infected cells but not in the endosome. Our findings reveal important features of the molecular basis of HPV infection, including the discovery that HPV uses different mechanisms to engage dynein at different times during entry, and identify potential targets for therapeutic approaches to inhibit HPV infection.</p><p><strong>Importance: </strong>Human papillomaviruses (HPVs) are small, non-enveloped DNA viruses that cause approximately 5% of human cancer. Like most other DNA viruses, HPV traffics to the nucleus during virus entry to successfully infect cells. We show here that HPV utilizes a cellular enzyme, Rab6a, during virus entry to engage the dynein molecular motor for transport along microtubules. Rab6a is required for complex formation between the HPV L2 capsid protein, dynein, and the dynein adaptor BICD2 in the <i>trans</i>-Golgi network (TGN). This complex is required for transport of the incoming virus out of the TGN as it journeys to the nucleus. Our findings identify potential targets for therapeutic approaches.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142469443","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}