scholarly journals Identification of spacer and protospacer sequence requirements in the Vibrio cholerae Type I-E CRISPR/Cas System

2020 ◽  
Author(s):  
Jacob Bourgeois ◽  
David W. Lazinski ◽  
Andrew Camilli
Keyword(s):  
Immune System ◽  
Vibrio Cholerae ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
Adaptive Immune System ◽  
Initial Study ◽  
Type I ◽  
Sequence Elements ◽  
Additional Sequence ◽  
Sequence Requirements

AbstractThe prokaryotic adaptive immune system CRISPR/Cas serves as defense against bacteriophage and invasive nucleic acid. A Type I-E CRISPR/Cas system has been detected in classical biotype isolates of Vibrio cholerae, the causative agent of the disease cholera. Experimental characterization of this system revealed a functional immune system that operates using a 5’-TT-3’ protospacer-adjacent motif (PAM) for interference. However, several designed spacers against the 5’-TT-3’ PAM do not interfere as expected, indicating further investigation of this system is necessary. In this study, we identified additional sequence requirements of a pyrimidine in the 5’ position of the spacer and purine in the complementary position of the protospacer using 873 unique spacers and 2267 protospacers mined from CRISPR arrays in deposited sequences of V. cholerae. We present bioinformatic evidence that during acquisition the protospacer purine is captured in the prespacer and that a 5’-RTT-3’ PAM is necessary for spacer acquisition. Finally, we demonstrate experimentally that a 5’-RTT-3’ PAM is necessary for CRISPR interference by designing and manipulating spacer and cognate PAMs in a plasmid conjugation assay and discover functional consequences of base pairing with the 5’ spacer pyrimidine in spacer efficacy.ImportanceBacterial CRISPR/Cas systems provide immunity by defending against phage and other invading elements. A thorough comprehension of the molecular mechanisms employed by these diverse systems will improve our understanding of bacteriophage-bacterial interactions and bacterial adaptation to foreign DNA. The Vibrio cholerae Type I-E system was previously identified in an extinct classical biotype and was partially characterized for its function. Here, using both bioinformatic and functional assays, we extend that initial study. We have found that the Type I-E system still exists in modern strains of V. cholerae. Furthermore, we defined additional sequence elements in both the CRISPR array and in target DNA that are required for immunity. CRISPR/Cas systems are now commonly used as precise and powerful genetic engineering tools. Knowledge of the sequences required for CRISPR/Cas immunity is a prerequisite for the effective design and experimental use of these systems. Our results greatly facilitate the effective use of one such system. Furthermore, we provide a publicly available script that assists in the detection and validation of CRISPR/Cas immunity requirements when such a system exists in any bacterial species.

scholarly journals Identification of Spacer and Protospacer Sequence Requirements in the Vibrio cholerae Type I-E CRISPR/Cas System

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Jacob Bourgeois ◽  
David W. Lazinski ◽  
Andrew Camilli
Keyword(s):  
Immune System ◽  
Vibrio Cholerae ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
Adaptive Immune System ◽  
Initial Study ◽  
Type I ◽  
Content Type ◽  
Protospacer Adjacent Motif ◽  
Sequence Elements

ABSTRACT The prokaryotic adaptive immune system CRISPR/Cas serves as a defense against bacteriophage and invasive nucleic acids. A type I-E CRISPR/Cas system has been detected in classical biotype isolates of Vibrio cholerae, the causative agent of the disease cholera. Experimental characterization of this system revealed a functional immune system that operates using a 5′-TT-3′ protospacer-adjacent motif (PAM) for interference. However, several designed spacers against the 5′-TT-3′ PAM do not interfere as expected, indicating that further investigation of this system is necessary. In this study, we identified additional conserved sequences, including a pyrimidine in the 5′ position of the spacer and a purine in the complementary position of the protospacer using 873 unique spacers and 2,267 protospacers mined from CRISPR arrays in deposited sequences of V. cholerae. We present bioinformatic evidence that during acquisition the protospacer purine is captured in the prespacer and that a 5′-RTT-3′ PAM is necessary for spacer acquisition. Finally, we demonstrate experimentally, by designing and manipulating spacer and cognate PAMs in a plasmid conjugation assay, that a 5′-RTT-3′ PAM is necessary for CRISPR interference, and we discover functional consequences for spacer efficacy related to the identity of the 5′ spacer pyrimidine. IMPORTANCE Bacterial CRISPR/Cas systems provide immunity by defending against phage and other invading elements. A thorough comprehension of the molecular mechanisms employed by these diverse systems will improve our understanding of bacteriophage-bacterium interactions and bacterial adaptation to foreign DNA. The Vibrio cholerae type I-E system was previously identified in an extinct classical biotype and was partially characterized for its function. Here, using both bioinformatic and functional assays, we extend that initial study. We have found that the type I-E system still exists in modern strains of V. cholerae. Furthermore, we defined additional sequence elements both in the CRISPR array and in target DNA that are required for immunity. CRISPR/Cas systems are now commonly used as precise and powerful genetic engineering tools. Knowledge of the sequences required for CRISPR/Cas immunity is a prerequisite for the effective design and experimental use of these systems. Our results greatly facilitate the effective use of one such system. Furthermore, we provide a publicly available software program that assists in the detection and validation of CRISPR/Cas immunity requirements when such a system exists in a bacterial species.

A Commitment to Lineage

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. SCI-22-SCI-22
Author(s):  
Laurie H. Glimcher ◽  
Vanja Lazarevic ◽  
Joerg Ermann ◽  
Wendy Garrett
Keyword(s):  
Colorectal Cancer ◽  
Immune System ◽  
Molecular Mechanisms ◽  
T Regulatory Cells ◽  
Inflammatory Diseases ◽  
Rectal Adenocarcinoma ◽  
Adaptive Immune System ◽  
T Helper ◽  
T Helper 1

Abstract Abstract SCI-22 The transcription factor T-bet, isolated in our laboratory a decade ago, is a master regulator of Type 1 immunity in cells of both the adaptive and innate immune system. In adaptive immunity, T-bet instigates genetic programs in T helper 1 (Th1) cells and is required for production of the hallmark Th1 cytokine IFNg. It simultaneously represses the differentiation of T helper 2 cells and the profibrotic cytokines IL-13 and TGFb. We have recently determined that T-bet is also a repressor of the Th17 genetic program and have established the molecular mechanisms that underpin that function. T-bet also controls the optimal differentiation and function of the cytolytic CD8 cell, and is required for the development of the natural killer T cell. T-bet deficient animals are largely protected from autoimmune/inflammatory diseases such as multiple sclerosis, systemic lupus, type 1 diabetes and inflammatory arthritis, but are susceptible to type 2 driven diseases such as asthma and scleroderma. An exception to this overall rule is our recent discovery that in the absence of an adaptive immune system, the majority of mice lacking T-bet develop a spontaneous ulcerative colitis that progresses to colonic dysplasia and rectal adenocarcinoma. This colitis and inflammation associated colorectal cancer are MyD88 independent, driven by colitogenic flora and ameliorated by treatment with TNF blockade, antibiotics, and transfer of T regulatory cells. This phenotype maps to the T-bet deficient dendritic cell that drives this pro-inflammatory program; selective over-expression of T-bet in DCs was sufficient to reduce colonic inflammation and prevent the progression to neoplasia. The molecular pathogenesis of TRUC colitis and colitis-associated colorectal (caCRC) shares several key features with human caCRC. This model of colitis and colitis-associated colorectal cancer provides opportunities to further understand host-microbial relationships in inflammation and neoplasia and test preventative and therapeutic strategies pre-clinically. The function and mechanism of action of T-bet in the pathogenesis of immune system driven diseases will be discussed. Disclosures: Glimcher: Merck: Consultancy, Patents & Royalties, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

scholarly journals Molecular Mechanisms of Leukocyte Migration and Its Potential Targeting—Lessons Learned From MKL1/SRF-Related Primary Immunodeficiency Diseases

2021 ◽  
Vol 12 ◽  
Author(s):  
Evelien G. G. Sprenkeler ◽  
Carla Guenther ◽  
Imrul Faisal ◽  
Taco W. Kuijpers ◽  
Susanna C. Fagerholm
Keyword(s):  
Immune System ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Serum Response Factor ◽  
Cell Function ◽  
Immune Cell ◽  
Leukocyte Adhesion ◽  
Adaptive Immune System ◽  
Lessons Learned ◽  
Megakaryoblastic Leukemia

Megakaryoblastic leukemia 1 (MKL1) deficiency is one of the most recently discovered primary immunodeficiencies (PIDs) caused by cytoskeletal abnormalities. These immunological “actinopathies” primarily affect hematopoietic cells, resulting in defects in both the innate immune system (phagocyte defects) and adaptive immune system (T-cell and B-cell defects). MKL1 is a transcriptional coactivator that operates together with serum response factor (SRF) to regulate gene transcription. The MKL/SRF pathway has been originally described to have important functions in actin regulation in cells. Recent results indicate that MKL1 also has very important roles in immune cells, and that MKL1 deficiency results in an immunodeficiency affecting the migration and function of primarily myeloid cells such as neutrophils. Interestingly, several actinopathies are caused by mutations in genes which are recognized MKL(1/2)-dependent SRF-target genes, namely ACTB, WIPF1, WDR1, and MSN. Here we summarize these and related (ARPC1B) actinopathies and their effects on immune cell function, especially focusing on their effects on leukocyte adhesion and migration. Furthermore, we summarize recent therapeutic efforts targeting the MKL/SRF pathway in disease.

scholarly journals Effects of Omega-3 Fatty Acids on Immune Cells

2019 ◽  
Vol 20 (20) ◽  
pp. 5028 ◽  
Author(s):  
Saray Gutiérrez ◽  
Sara L Svahn ◽  
Maria E Johansson
Keyword(s):  
Fatty Acids ◽  
Immune System ◽  
Immune Cells ◽  
Molecular Mechanisms ◽  
Adaptive Immune System ◽  
Cellular Membrane ◽  
Membrane Properties ◽  
Omega 3 Fatty Acids ◽  
Omega 3 ◽  
Adaptive Immune

Alterations on the immune system caused by omega-3 fatty acids have been described for 30 years. This family of polyunsaturated fatty acids exerts major alterations on the activation of cells from both the innate and the adaptive immune system, although the mechanisms for such regulation are diverse. First, as a constitutive part of the cellular membrane, omega-3 fatty acids can regulate cellular membrane properties, such as membrane fluidity or complex assembly in lipid rafts. In recent years, however, a new role for omega-3 fatty acids and their derivatives as signaling molecules has emerged. In this review, we describe the latest findings describing the effects of omega-3 fatty acids on different cells from the immune system and their possible molecular mechanisms.

CRISPR/Cas-Systems: characteristics and possibilities of use for editing bacterial genomes

Bacteriology ◽  
2020 ◽  
Vol 5 (2) ◽  
pp. 38-48
Author(s):  
I.A. Blatov ◽  
◽  
A.S. Shchurova ◽  
D.Yu. Guschin ◽  
S.D. Zvereva ◽  
...  
Keyword(s):  
Immune System ◽  
Genome Editing ◽  
Bacterial Species ◽  
Adaptive Immune System ◽  
Bacterial Genomes ◽  
Modern Biology ◽  
Adaptive Immune ◽  
History Of ◽  
Genomic Editing ◽  
Classification Structure

CRISPR-Cas is the adaptive immune system of bacteria and archaea. Since 2012, when the first opportunity to use the CRISPR/Cas system for genome editing was realized, the number of studies in this area has been growing rapidly. Today, genomic editing to modify specific regions of the genomes of various organisms is considered one of the key methodologies of modern biology. This review is devoted to the history of discovery, classification, structure, operational mechanisms of CRISPRCas systems and strategies for editing the genomes of various bacterial species using this technology. Key words: genome editing, genome, CRISPR-Cas system, bacteria

SFRP5 Inhibits Early Stages Of B-Cell Differentiation and Modulates Estrogen-Related Changes In The Immune System

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2422-2422
Author(s):  
Takafumi Yokota ◽  
Kenji Oritani ◽  
Takao Sudo ◽  
Tomohiko Ishibashi ◽  
Yukiko Doi ◽  
...  
Keyword(s):  
Immune System ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Molecular Mechanisms ◽  
Adaptive Immune System ◽  
B Cell Differentiation ◽  
B Lymphopoiesis

Abstract A large body of research has demonstrated that the maternal immune system is elaborately regulated during pregnancy to establish immunological tolerance to the fetus. Although our previous works have revealed that female sex hormones, particularly estrogen, play pivotal roles in suppressing maternal B-lymphopoiesis, the precise molecular mechanisms that mediate their functions are largely unknown. Because T and B lymphocytes function coordinately in the adaptive immune system, the inhibition of B-lymphopoiesis during pregnancy should be involved, at least in part, in “maternal-fetal immune tolerance.” Understanding the molecular mechanisms of tolerance would contribute to the development of new methods to inhibit immune responses after organ transplantation, such as rejection by the host or graft-versus-host diseases. The goal of our present study is to identify the molecular pathways through which estrogen exerts its suppressive effect on B-lymphopoiesis. We performed global analyses of estrogen-inducible genes in bone marrow (BM) stromal cells and identified the secreted frizzled-related protein (sFRP) family. A sFRP1-immunoglobulin G (Ig) fusion protein inhibited early differentiation of B-cells originating from BM-derived hematopoietic stem/progenitor cells (HSPC) in culture (Yokota T. et al. Journal of Immunol, 2008). Conversely, sFRP1 deficiency in vivo caused dysregulation of HSPC homeostasis in BM and aberrant increase of peripheral B lymphocytes (Renström J. et al. Cell Stem Cell, 2009). Therefore, in the present study we generated sFRP1 transgenic chimera (TC) mice that produced high levels of circulating sFRP1 after birth to examine the influence of sFRP1 on adult lymphopoiesis in vivo. Further, we generated sFRP5 TC mice using the same procedure to determine whether there were functional differences or redundancies between sFRP1 and sFRP5. The two are most closely related isoforms among the sFRP family and are known to play redundant roles during embryonic development; however, their physiological function in the immune system is largely unknown. Unexpectedly, while only subtle change was detected in the lymphoid lineage of sFRP1 TC mice, we found that the number of B cells was significantly reduced in the sFRP5 TC mice. The frequency of B cells, which normally account for approximately 50% of peripheral leukocytes of wild-type (WT) mice, was reduced to less than 20% in the sFRP5 TC mice. The suppression was likely specific to the B lineage, because overexpression of sFRP5 did not affect myeloid, T, or NK cells. Compared with WT littermates, the body size of sFRP5 TC mice was slightly, but significantly smaller. Thymocyte counts were not affected. In contrast, the number of splenocytes, particularly those of the B lineage, significantly decreased. In BM of sFRP5 TC mice, early B-cell differentiation was inhibited, resulting in the accumulation of cells whose phenotype corresponds to those of common lymphoid progenitors (CLPs). Gene array analyses of the accumulated CLPs indicated that sFRP5 affects the expression of adaptive immune system-related genes. Further, the sFRP5 overexpression was found to induce the expression of Wnt and Notch-related molecules that regulate the integrity of HSPCs. To determine the physiological involvement of sFRP5 in the inhibition of early B-cell differentiation, we exploited mice lacking sFRP5. It is noteworthy that, although the level of sFRP5 expression was minimal in steady-state BM, it was markedly induced after estrogen treatment. We injected water-soluble β-estradiol into WT or sFRP5-null mice for 4 days and evaluated their lympho-hematopoiesis 12 h after the last injection. While the highly HSPC-enriched Lineage- Sca-1+ c-kitHi Flt3- fraction of WT mice was resistant to the treatment, the same fraction of sFRP5-null mice showed a declining trend. Further, although the CLP fraction was significantly reduced in both strains, CLPs of sFRP5-null mice were more sensitive to estrogen than those of WT. We also performed gene expression analyses of WT and sFRP5-null mice after the estrogen treatment. We found that estrogen induced the expression of Hes1 in HSPCs of WT but not sFRP5-null mice. Thus, we conclude that estrogen-inducible sFRP5 blocks the differentiation of HSPCs in BM to B-lymphocytes in the presence of high levels of estrogen, at least in part by activation of the Notch pathway. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Defective innate immunity predisposes murine neonates to poor sepsis outcome but is reversed by TLR agonists

Blood ◽  
2008 ◽  
Vol 112 (5) ◽  
pp. 1750-1758 ◽  
Author(s):  
James L. Wynn ◽  
Philip O. Scumpia ◽  
Robert D. Winfield ◽  
Matthew J. Delano ◽  
Kindra Kelly-Scumpia ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Immune System ◽  
Adaptive Immune System ◽  
Neutrophil Recruitment ◽  
Polymicrobial Sepsis ◽  
Type I ◽  
Sepsis Mortality ◽  
Tlr Agonists ◽  
Adaptive Immune ◽  
Increased Risk

Abstract Neonates exhibit an increased risk of sepsis mortality compared with adults. We show that in contrast to adults, survival from polymicrobial sepsis in murine neonates does not depend on an intact adaptive immune system and is not improved by T cell–directed adaptive immunotherapy. Furthermore, neonates manifest an attenuated inflammatory and innate response to sepsis, and have functional defects in their peritoneal CD11b+ cells. Activation of innate immunity with either a Toll-like receptor 4 (TLR4) or TLR7/8 agonist, but not a TLR3 agonist, increased the magnitude, but abbreviated the early systemic inflammatory response, reduced bacteremia, and improved survival to polymicrobial sepsis. TLR4 agonist pretreatment enhanced peritoneal neutrophil recruitment with increased oxidative burst production, whereas the TLR7/8 agonist also enhanced peritoneal neutrophil recruitment with increased phagocytic ability. These benefits were independent of the adaptive immune system and type I interferon signaling. Improving innate immune function with select TLR agonists may be a useful strategy to prevent neonatal sepsis mortality.

scholarly journals RAGE and TLRs as Key Targets for Antiatherosclerotic Therapy

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Wioletta Olejarz ◽  
Dominika Łacheta ◽  
Alicja Głuszko ◽  
Ewa Migacz ◽  
Wojciech Kukwa ◽  
...  
Keyword(s):  
Immune System ◽  
Inflammatory Responses ◽  
Adaptive Immune System ◽  
Primary Response ◽  
Type I ◽  
Adaptive Responses ◽  
Effector Cells ◽  
Antiatherosclerotic Therapy ◽  
Molecular Patterns ◽  
Direct Killing

Receptor for advanced glycation end-products (RAGE) and toll-like receptors (TLRs) are the key factors indicating a danger to the organism. They recognize the microbial origin pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). The primary response induced by PAMPs or DAMPs is inflammation. Excessive stimulation of the innate immune system occurs in arterial wall with the participation of effector cells. Persistent adaptive responses can also cause tissue damage and disease. However, inflammation mediated by the molecules innate responses is an important way in which the adaptive immune system protects us from infection. The specific detection of PAMPs and DAMPs by host receptors drives a cascade of signaling that converges at nuclear factor-κB (NF-κB) and interferon regulatory factors (IRFs) and induces the secretion of proinflammatory cytokines, type I interferon (IFN), and chemokines, which promote direct killing of the pathogen. Therefore, signaling of these receptors’ pathways also appear to present new avenue for the modulation of inflammatory responses and to serve as potential novel therapeutic targets for antiatherosclerotic therapy.

scholarly journals Human Type I Interferon Antiviral Effects in Respiratory and Reemerging Viral Infections

2020 ◽  
Vol 2020 ◽  
pp. 1-27 ◽  
Author(s):  
Patricio L. Acosta ◽  
Alana B. Byrne ◽  
Diego R. Hijano ◽  
Laura B. Talarico
Keyword(s):  
Immune System ◽  
Host Defense ◽  
Immune Modulation ◽  
Molecular Mechanisms ◽  
Viral Infections ◽  
Current Knowledge ◽  
Type I Interferons ◽  
Type I ◽  
Antiviral Effects ◽  
Wide Range

Type I interferons (IFN-I) are a group of related proteins that help regulate the activity of the immune system and play a key role in host defense against viral infections. Upon infection, the IFN-I are rapidly secreted and induce a wide range of effects that not only act upon innate immune cells but also modulate the adaptive immune system. While IFN-I and many IFN stimulated genes are well-known for their protective antiviral role, recent studies have associated them with potential pathogenic functions. In this review, we summarize the current knowledge regarding the complex effects of human IFN-I responses in respiratory as well as reemerging flavivirus infections of public health significance and the molecular mechanisms by which viral proteins antagonize the establishment of an antiviral host defense. Antiviral effects and immune modulation of IFN-stimulated genes is discussed in resisting and controlling pathogens. Understanding the mechanisms of these processes will be crucial in determining how viral replication can be effectively controlled and in developing safe and effective vaccines and novel therapeutic strategies.

SLPI and elafin: one glove, many fingers

2005 ◽  
Vol 110 (1) ◽  
pp. 21-35 ◽  
Author(s):  
Steven E. Williams ◽  
Thomas I. Brown ◽  
Ali Roghanian ◽  
Jean-Michel Sallenave
Keyword(s):  
Immune Response ◽  
Immune System ◽  
Immune Responses ◽  
Adaptive Immune Response ◽  
Adaptive Immune System ◽  
Innate Immune ◽  
Type I ◽  
Adaptive Immune ◽  
Pathological Conditions ◽  
Related Proteins

Elafin and SLPI (secretory leucocyte protease inhibitor) have multiple important roles both in normal homoeostasis and at sites of inflammation. These include antiprotease and antimicrobial activity as well as modulation of the response to LPS (lipopolysaccharide) stimulation. Elafin and SLPI are members of larger families of proteins secreted predominantly at mucosal sites, and have been shown to be modulated in multiple pathological conditions. We believe that elafin and SLPI are important molecules in the controlled functioning of the innate immune system, and may have further importance in the integration of this system with the adaptive immune response. Recent interest has focused on the influence of inflamed tissues on the recruitment and phenotypic modulation of cells of the adaptive immune system and, indeed, the local production of elafin and SLPI indicate that they are ideally placed in this regard. Functionally related proteins, such as the defensins and cathelicidins, have been shown to have direct effects upon dendritic cells with potential alteration of their phenotype towards type I or II immune responses. This review addresses the multiple functions of elafin and SLPI in the inflammatory response and discusses further their roles in the development of the adaptive immune response.

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