scholarly journals Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: Insights from a natural arms-race

2018 ◽  
Author(s):  
Amelia C. McKitterick ◽  
Kristen N. LeGault ◽  
Angus Angermeyer ◽  
Muniral Alam ◽  
Kimberley D. Seed
Keyword(s):  
High Throughput Sequencing ◽  
Mobile Genetic Element ◽  
Arms Race ◽  
Type I ◽  
Genetic Element ◽  
Wild Populations ◽  
Adaptive Immune ◽  
Adaptive Immune Systems ◽  
Phage Activity

AbstractCRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defense against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae. Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not equally interfere with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses.

scholarly journals Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: insights from a natural arms race

2019 ◽  
Vol 374 (1772) ◽  
pp. 20180089 ◽  
Author(s):  
Amelia C. McKitterick ◽  
Kristen N. LeGault ◽  
Angus Angermeyer ◽  
Munirul Alam ◽  
Kimberley D. Seed
Keyword(s):  
High Throughput Sequencing ◽  
Mobile Genetic Element ◽  
Arms Race ◽  
Type I ◽  
Helicase Activity ◽  
Wild Populations ◽  
Immune Systems ◽  
Adaptive Immune ◽  
Adaptive Immune Systems

CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defence against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae . Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not interfere equally with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and is dependent on Cas2-3 helicase activity. We propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory-acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.

scholarly journals IN LABORATORY GENERATION AND MATURATION OF HUMAN MONOCYTE-DERIVED DENDRITIC CELLS FOR CANCER IMMUNOTHERAPY

2020 ◽  
pp. 46-52
Author(s):  
KANCHAN K. MISHRA ◽  
SUMIT BHARADVA ◽  
MEGHNAD G. JOSHI ◽  
ARVIND GULBAKE
Keyword(s):  
Dendritic Cells ◽  
Immune Responses ◽  
Gradient Centrifugation ◽  
Critical Role ◽  
Human Monocyte ◽  
Blood Monocytes ◽  
Adaptive Immune ◽  
Immunodeficiency Virus ◽  
Adaptive Immune Systems

Dendritic cells (DCs) play a critical role in the regulation of adaptive immune responses, furthermore they act as a bridge between the innate and the adaptive immune systems they have been ideal candidates for cell-based immunotherapy of cancers and infections in humans. The first reported trial using DCs in 1995, since they have been used in trials all over the world for several of indications, including cancer and human immunodeficiency virus infection. Generally, for in vitro experiments or for DCs vaccination monocyte-derived dendritic cells (moDCs) were generated from purified monocytes that isolated from peripheral blood by density gradient centrifugation. A variety of methods can be used for enrichment of monocytes for generation of clinical-grade DCs. Herein we summarized up to date understanding of systems and inputs used in procedures to differentiate DCs from blood monocytes in vitro.

scholarly journals CRISPR-Cas immunity repressed by a biofilm-activating pathway inPseudomonas aeruginosa

2019 ◽  
Author(s):  
Adair L. Borges ◽  
Bardo Castro ◽  
Sutharsan Govindarajan ◽  
Tina Solvik ◽  
Veronica Escalante ◽  
...  
Keyword(s):  
Genetic Screen ◽  
Type I ◽  
Two Component System ◽  
Component System ◽  
Forward Genetic Screen ◽  
Immune Systems ◽  
Adaptive Immune ◽  
Biofilm Production ◽  
Two Component ◽  
Adaptive Immune Systems

CRISPR-Cas systems are adaptive immune systems that protect bacteria from bacteriophage (phage) infection. To provide immunity, RNA-guided protein surveillance complexes recognize foreign nucleic acids, triggering their destruction by Cas nucleases. While the essential requirements for immune activity are well understood, the physiological cues that regulate CRISPR-Cas expression are not. Here, a forward genetic screen identifies a two-component system (KinB/AlgB), previously characterized in regulatingPseudomonas aeruginosavirulence and biofilm establishment, as a regulator of the biogenesis and activity of the Type I-F CRISPR-Cas system. Downstream of the KinB/AlgB system, activators of biofilm production AlgU (a σEorthologue) and AlgR, act as repressors of CRISPR-Cas activity during planktonic and surface-associated growth. AmrZ, another biofilm activator, functions as a surface-specific repressor of CRISPR-Cas immunity.Pseudomonasphages and plasmids have taken advantage of this regulatory scheme, and carry hijacked homologs of AmrZ, which are functional CRISPR-Cas repressors. This suggests that while CRISPR-Cas regulation may be important to limit self-toxicity, endogenous repressive pathways represent a vulnerability for parasite manipulation.

scholarly journals Structural basis for inhibition of the type I-F CRISPR–Cas surveillance complex by AcrIF4, AcrIF7 and AcrIF14

2020 ◽  
Author(s):  
Clinton Gabel ◽  
Zhuang Li ◽  
Heng Zhang ◽  
Leifu Chang
Keyword(s):  
Genome Editing ◽  
Conformational Changes ◽  
Structural Basis ◽  
Type I ◽  
Structural Detail ◽  
Immune Systems ◽  
Helical Bundle ◽  
Adaptive Immune ◽  
Target Dna ◽  
Adaptive Immune Systems

Abstract CRISPR–Cas systems are adaptive immune systems in bacteria and archaea to defend against mobile genetic elements (MGEs) and have been repurposed as genome editing tools. Anti-CRISPR (Acr) proteins are produced by MGEs to counteract CRISPR–Cas systems and can be used to regulate genome editing by CRISPR techniques. Here, we report the cryo-EM structures of three type I-F Acr proteins, AcrIF4, AcrIF7 and AcrIF14, bound to the type I-F CRISPR–Cas surveillance complex (the Csy complex) from Pseudomonas aeruginosa. AcrIF4 binds to an unprecedented site on the C-terminal helical bundle of Cas8f subunit, precluding conformational changes required for activation of the Csy complex. AcrIF7 mimics the PAM duplex of target DNA and is bound to the N-terminal DNA vise of Cas8f. Two copies of AcrIF14 bind to the thumb domains of Cas7.4f and Cas7.6f, preventing hybridization between target DNA and the crRNA. Our results reveal structural detail of three AcrIF proteins, each binding to a different site on the Csy complex for inhibiting degradation of MGEs.

scholarly journals Detection of virulence associated genes, haemolysin and protease amongst Vibrio cholerae isolated in Malaysia

2000 ◽  
Vol 125 (1) ◽  
pp. 27-34 ◽  
Author(s):  
L. IYER ◽  
J. VADIVELU ◽  
S. D. PUTHUCHEARY
Keyword(s):  
Vibrio Cholerae ◽  
Dna Hybridization ◽  
Type I ◽  
Genetic Element ◽  
Pathogenic Potential ◽  
Colonization Factor ◽  
Agar Diffusion Assay ◽  
Phage Receptor ◽  
Diffusion Assay

Eighty-four strains of Vibrio cholerae O1, O139 and non-O1/non-O139 from clinical and environmental sources were investigated for the presence of the toxin co-regulated pilus gene, tcpA, the virulence cassette genes ctxA, zot, ace and cep and also for their ability to elaborate haemolysin and protease. The ctxA and zot genes were detected using DNA–DNA hybridization while the ace, cep and tcpA genes were detected using PCR. Production of haemolysin and protease was detected using mammalian erythrocytes and an agar diffusion assay respectively. Analysis of their virulence profiles showed six different groups designated Type I to Type VI and the major distinguishing factor among these profiles was in the in vitro production of haemolysin and/or protease. Clinical O1, O139 and environmental O1 strains were similar with regard to presence of the virulence cassette genes. All environmental O1 strains with the exception of one were found to possess ctxA, zot and ace giving rise to the probability that these strains may actually be of clinical origin. One strain which had only cep but none of the toxin genes may be a true environmental isolate. The virulence cassette and colonization factor genes were absent in all non-O1/non-O139 environmental strains but production of both the haemolysin and protease was present, indicating that these may be putative virulence factors. These findings suggest that with regard to its pathogenic potential, only strains of the O1 and O139 serogroup that possess the tcpA gene which encodes the phage receptor, have the potential to acquire the CTX genetic element and become choleragenic.

scholarly journals Functional Exhaustion of Type I and II Interferons Production in Severe COVID-19 Patients

2021 ◽  
Vol 7 ◽  
Author(s):  
Caroline Ruetsch ◽  
Vesna Brglez ◽  
Marion Crémoni ◽  
Kévin Zorzi ◽  
Céline Fernandez ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Immune Cells ◽  
Type I ◽  
Type Ii ◽  
Systemic Complications ◽  
Adaptive Immune ◽  
Functional Exhaustion ◽  
Cytokine Levels ◽  
Stimulation Of

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged in Wuhan in December 2019 and has since spread across the world. Even though the majority of patients remain completely asymptomatic, some develop severe systemic complications. In this prospective study we compared the immunological profile of 101 COVID-19 patients with either mild, moderate or severe form of the disease according to the WHO classification, as well as of 50 healthy subjects, in order to identify functional immune factors independently associated with severe forms of COVID-19. Plasma cytokine levels, and cytokine levels upon in vitro non-specific stimulation of innate and adaptive immune cells, were measured at several time points during the course of the disease. As described previously, inflammatory cytokines IL1β, IL6, IL8, and TNFα associated with cytokine storm were significantly increased in the plasma of moderate and severe COVID-19 patients (p < 0.0001 for all cytokines). During follow-up, plasma IL6 levels decreased between the moment of admission to the hospital and at the last observation carried forward for patients with favorable outcome (p = 0.02148). After in vitro stimulation of immune cells from COVID-19 patients, reduced levels of both type I and type II interferons (IFNs) upon in vitro stimulation were correlated with increased disease severity [type I IFN (IFNα): p > 0.0001 mild vs. moderate and severe; type II IFN (IFNγ): p = 0.0002 mild vs. moderate and p < 0.0001 mild vs. severe] suggesting a functional exhaustion of IFNs production. Stimulated IFNα levels lower than 2.1 pg/ml and IFNγ levels lower than 15 IU/mL at admission to the hospital were associated with more complications during hospitalization (p = 0.0098 and p =0.0002, respectively). A low IFNγ level was also confirmed by multivariable analysis [p = 0.0349 OR = 0.98 (0.962; 0.999)] as an independent factor of complications. In vitro treatment with type IFNα restored type IFNγ secretion in COVID-19 patients while the secretion of pro-inflammatory cytokines IL6 and IL1β remained stable or decreased, respectively. These results (a) demonstrate a functional exhaustion of both innate and adaptive immune response in severe forms of COVID-19; (b) identify IFNα and IFNγ as new potential biomarkers of severity; and (c) highlight the importance of targeting IFNs when considering COVID-19 treatment in order to re-establish a normal balance between inflammatory and Th1 effector cytokines.

scholarly journals Role of Type I Interferons on Filovirus Pathogenesis

Vaccines ◽  
2019 ◽  
Vol 7 (1) ◽  
pp. 22 ◽  
Author(s):  
Beatriz Escudero-Pérez ◽  
César Muñoz-Fontela
Keyword(s):  
Animal Models ◽  
Immune Responses ◽  
Marburg Virus ◽  
Type I Interferons ◽  
Type I ◽  
Adaptive Immune ◽  
Antigen Presenting

Filoviruses, such as Ebola and Marburg virus, encode viral proteins with the ability to counteract the type I interferon (IFN-I) response. These IFN-I antagonist proteins are crucial to ensure virus replication, prevent an antiviral state in infected and bystander cells, and impair the ability of antigen-presenting cells to initiate adaptive immune responses. However, in recent years, a number of studies have underscored the conflicting data between in vitro studies and in vivo data obtained in animal models and clinical studies during outbreaks. This review aims to summarize these data and to discuss the relative contributions of IFN-α and IFN-β to filovirus pathogenesis in animal models and humans. Finally, we evaluate the putative utilization of IFN-I in post-exposure therapy and its implications as a biomarker of vaccine efficacy.

In vitro immunogenicity prediction: bridging between innate and adaptive immunity

Bioanalysis ◽  
2021 ◽  
Author(s):  
Sivan Cohen ◽  
Shan Chung
Keyword(s):  
Immune Cells ◽  
Immune Cell ◽  
In Vitro Assays ◽  
Adaptive Immune ◽  
Adaptive Immune Cells ◽  
Clinical Consequences ◽  
Immune Cell Response ◽  
Adaptive Immune Systems

Development of antidrug antibodies (ADAs) is an undesirable potential outcome of administration of biotherapeutics and involves the innate and adaptive immune systems. ADAs can have detrimental clinical consequences: they can reduce biotherapeutic efficacy or produce adverse events. Because animal models are considered poor predictors of immunogenicity in humans, in vitro assays with human innate and adaptive immune cells are commonly used alternatives that can reveal cell-mediated unwanted immune responses. Multiple methods have been developed to assess the immune cell response following exposure to biotherapeutics and estimate the potential immunogenicity of biotherapeutics. This review highlights the role of innate and adaptive immune cells as the drivers of immunogenicity and summarizes the use of these cells in assays to predict clinical ADA.

scholarly journals Why put up with immunity when there is resistance: an excursion into the population and evolutionary dynamics of restriction–modification and CRISPR-Cas

2019 ◽  
Vol 374 (1772) ◽  
pp. 20180096 ◽  
Author(s):  
James Gurney ◽  
Maroš Pleška ◽  
Bruce R. Levin
Keyword(s):  
Evolutionary Dynamics ◽  
Arms Race ◽  
Lytic Phage ◽  
Bacterial Populations ◽  
Immune Systems ◽  
Adaptive Immune ◽  
Escape Mutants ◽  
Adaptive Immune Systems ◽  
Restriction Modification ◽  
Coevolutionary Arms Race

Bacteria can readily generate mutations that prevent bacteriophage (phage) adsorption and thus make bacteria resistant to infections with these viruses. Nevertheless, the majority of bacteria carry complex innate and/or adaptive immune systems: restriction–modification (RM) and CRISPR-Cas, respectively. Both RM and CRISPR-Cas are commonly assumed to have evolved and be maintained to protect bacteria from succumbing to infections with lytic phage. Using mathematical models and computer simulations, we explore the conditions under which selection mediated by lytic phage will favour such complex innate and adaptive immune systems, as opposed to simple envelope resistance. The results of our analysis suggest that when populations of bacteria are confronted with lytic phage: (i) In the absence of immunity, resistance to even multiple bacteriophage species with independent receptors can evolve readily. (ii) RM immunity can benefit bacteria by preventing phage from invading established bacterial populations and particularly so when there are multiple bacteriophage species adsorbing to different receptors. (iii) Whether CRISPR-Cas immunity will prevail over envelope resistance depends critically on the number of steps in the coevolutionary arms race between the bacteria-acquiring spacers and the phage-generating CRISPR-escape mutants. We discuss the implications of these results in the context of the evolution and maintenance of RM and CRISPR-Cas and highlight fundamental questions that remain unanswered. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.

scholarly journals Mesenchymal Stem Cells Regulate the Innate and Adaptive Immune Responses Dampening Arthritis Progression

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
R. A. Contreras ◽  
F. E. Figueroa ◽  
F. Djouad ◽  
P. Luz-Crawford
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Therapeutic Potential ◽  
Multipotent Stem Cells ◽  
Immune Systems ◽  
Adaptive Immune ◽  
Adaptive Immune Systems

Mesenchymal stem cells (MSCs) are multipotent stem cells that are able to immunomodulate cells from both the innate and the adaptive immune systems promoting an anti-inflammatory environment. During the last decade, MSCs have been intensively studiedin vitroandin vivoin experimental animal model of autoimmune and inflammatory disorders. Based on these studies, MSCs are currently widely used for the treatment of autoimmune diseases such as rheumatoid arthritis (RA) characterized by complex deregulation of the immune systems. However, the therapeutic properties of MSCs in arthritis are still controverted. These controversies might be due to the diversity of MSC sources and isolation protocols used, the time, the route and dose of MSC administration, the variety of the mechanisms involved in the MSCs suppressive effects, and the complexity of arthritis pathogenesis. In this review, we discuss the role of the interactions between MSCs and the different immune cells associated with arthritis pathogenesis and the possible means described in the literature that could enhance MSCs therapeutic potential counteracting arthritis development and progression.

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