Eukaryotic Immune Systems Research Articles

The Prokaryotic Roots of Eukaryotic Immune Systems.

Over the past two decades, studies have revealed profound evolutionary connections between prokaryotic and eukaryotic immune systems, challenging the notion of their unrelatedness. Immune systems across the tree of life share an operational framework, shaping their biochemical logic and evolutionary trajectories. The diversification of immune genes in the prokaryotic superkingdoms, followed by lateral transfer to eukaryotes, was central to the emergence of innate immunity in the latter. These include protein domains related to nucleotide second messenger-dependent systems, NAD+/nucleotide degradation, and P-loop NTPase domains of the STAND and GTPase clades playing pivotal roles in eukaryotic immunity and inflammation. Moreover, several domains orchestrating programmed cell death, ultimately of prokaryotic provenance, suggest an intimate link between immunity and the emergence of multicellularity in eukaryotes such as animals. While eukaryotes directly adopted some proteins from bacterial immune systems, they repurposed others for new immune functions from bacterial interorganismal conflict systems. These emerging immune components hold substantial biotechnological potential.

Functionally comparable but evolutionarily distinct nucleotide-targetingeffectors help identify conserved paradigms across diverse immune systems.

While nucleic acid-targeting effectors are known to be central to biological conflicts and anti-selfish element immunity, recent findings have revealed immune effectors that target their building blocks and the cellular energy currency-free nucleotides. Through comparative genomics and sequence-structure analysis, we identified several distinct effector domains, which we named Calcineurin-CE, HD-CE, and PRTase-CE. These domains, along with specific versions of the ParB and MazG domains, are widely present in diverse prokaryotic immune systems and are predicted to degrade nucleotides by targeting phosphate or glycosidic linkages. Our findings unveil multiple potential immune systems associated with at least 17 different functional themes featuring these effectors. Some of these systems sense modified DNA/nucleotides from phages or operate downstream of novel enzymes generating signaling nucleotides. We also uncovered a class of systems utilizing HSP90- and HSP70-related modules as analogs of STAND and GTPase domains that are coupled to these nucleotide-targeting- or proteolysis-induced complex-forming effectors. While widespread in bacteria, only a limited subset of nucleotide-targeting effectors was integrated into eukaryotic immune systems, suggesting barriers to interoperability across subcellular contexts. This work establishes nucleotide-degrading effectors as an emerging immune paradigm and traces their origins back to homologous domains in housekeeping systems.

Bacterial NLR-related proteins protect against phage

Bacteria use a wide range of immune pathways to counter phage infection. A subset of these genes shares homology with components of eukaryotic immune systems, suggesting that eukaryotes horizontally acquired certain innate immune genes from bacteria. Here, we show that proteins containing a NACHT module, the central feature of the animal nucleotide-binding domain and leucine-rich repeat containing gene family (NLRs), are found in bacteria and defend against phages. NACHT proteins are widespread in bacteria, provide immunity against both DNA and RNA phages, and display the characteristic C-terminal sensor, central NACHT, and N-terminal effector modules. Some bacterial NACHT proteins have domain architectures similar to the human NLRs that are critical components of inflammasomes. Human disease-associated NLR mutations that cause stimulus-independent activation of the inflammasome also activate bacterial NACHT proteins, supporting a shared signaling mechanism. This work establishes that NACHT module-containing proteins are ancient mediators of innate immunity across the tree of life.

Pangenomic analysis reveals plant NAD+ manipulation as an important virulence activity of bacterial pathogen effectors

Nicotinamide adenine dinucleotide (NAD+) has emerged as a key component in prokaryotic and eukaryotic immune systems. The recent discovery that Toll/interleukin-1 receptor (TIR) proteins function as NAD+ hydrolases (NADase) links NAD+-derived small molecules with immune signaling. We investigated pathogen manipulation of host NAD+ metabolism as a virulence strategy. Using the pangenome of the model bacterial pathogen Pseudomonas syringae, we conducted a structure-based similarity search from 35,000 orthogroups for type III effectors (T3Es) with potential NADase activity. Thirteen T3Es, including five newly identified candidates, were identified that possess domain(s) characteristic of seven NAD+-hydrolyzing enzyme families. Most Pseudomonas syringae strains that depend on the type III secretion system to cause disease, encode at least one NAD+-manipulating T3E, and many have several. We experimentally confirmed the type III-dependent secretion of a novel T3E, named HopBY, which shows structural similarity to both TIR and adenosine diphosphate ribose (ADPR) cyclase. Homologs of HopBY were predicted to be type VI effectors in diverse bacterial species, indicating potential recruitment of this activity by microbial proteins secreted during various interspecies interactions. HopBY efficiently hydrolyzes NAD+ and specifically produces 2'cADPR, which can also be produced by TIR immune receptors of plants and by other bacteria. Intriguingly, this effector promoted bacterial virulence, indicating that 2'cADPR may not be the signaling molecule that directly initiates immunity. This study highlights a host-pathogen battleground centered around NAD+ metabolism and provides insight into the NAD+-derived molecules involved in plant immunity.

Plant Antimicrobial Peptides as Potential Tool for Topic Treatment of Hidradenitis Suppurativa.

Among chronic skin autoinflammatory diseases, Hidradenitis Suppurativa (HS) stands out for its chronicity, highly variable condition, and profound impact on the patients’ quality of life. HS is characterized by suppurative skin lesions in diverse body areas, including deep-seated painful nodules, abscesses, draining sinus, and bridged scars, among others, with typical topography. To date, HS is considered a refractory disease and medical treatments aim to reduce the incidence, the infection, and the pain of the lesions. For this purpose, different classes of drugs, including anti-inflammatory molecules, antibiotics and biological drugs are being used. Antimicrobial peptides (AMPs), also called defense peptides, emerge as a new class of therapeutic compounds, with broad-spectrum antimicrobial action, in addition to reports on their anti-inflammatory, healing, and immunomodulating activity. Such peptides are present in prokaryotes and eukaryotes, as part of the innate eukaryotic immune system. It has been proposed that a deregulation in the expression of AMPs in human epithelial tissues of HS patients may be associated with the etiology of this skin disease. In this scenario, plant AMPs stand out for their richness, diversity of types, and broad antimicrobial effects, with potential application for topical systemic use in patients affected by HS.

The small molecule Zaractin activates ZAR1-mediated immunity in Arabidopsis

Pathogenic effector proteins use a variety of enzymatic activities to manipulate host cellular proteins and favor the infection process. However, these perturbations can be sensed by nucleotide-binding leucine-rich-repeat (NLR) proteins to activate effector-triggered immunity (ETI). Here we have identified a small molecule (Zaractin) that mimics the immune eliciting activity of the Pseudomonas syringae type III secreted effector (T3SE) HopF1r and show that both HopF1r and Zaractin activate the same NLR-mediated immune pathway in Arabidopsis Our results demonstrate that the ETI-inducing action of pathogenic effectors can be harnessed to identify synthetic activators of the eukaryotic immune system.

MagC is a NplC/P60‐like member of the α‐2‐macroglobulin Mag complex of Pseudomonas aeruginosa that interacts with peptidoglycan

Bacterial α-2 macroglobulins (A2Ms) structurally resemble the large spectrum protease inhibitors of the eukaryotic immune system. In Pseudomonasaeruginosa, MagD acts as an A2M and is expressed within a six-gene operon encoding the MagA-F proteins. In this work, we employ isothermal calorimetry (ITC), analytical ultracentrifugation (AUC), and X-ray crystallography to investigate the function of MagC and show that MagC associates with the macroglobulin complex and with the peptidoglycan (PG). However, the catalytic residues of MagC display an inactive conformation that could suggest that it binds to PG but does not degrade it. We hypothesize that MagC could serve as an anchor between the MagD macroglobulin and the PG and could provide stabilization and/or regulation for the entire complex.

Identification of Uncharacterized Components of Prokaryotic Immune Systems and Their Diverse Eukaryotic Reformulations.

Nucleotide-activated effector deployment, prototyped by interferon-dependent immunity, is a common mechanistic theme shared by immune systems of several animals and prokaryotes. Prokaryotic versions include CRISPR-Cas with the CRISPR polymerase domain, their minimal variants, and systems with second messenger oligonucleotide or dinucleotide synthetase (SMODS). Cyclic or linear oligonucleotide signals in these systems help set a threshold for the activation of potentially deleterious downstream effectors in response to invader detection. We establish such a regulatory mechanism to be a more general principle of immune systems, which can also operate independently of such messengers. Using sensitive sequence analysis and comparative genomics, we identify 12 new prokaryotic immune systems, which we unify by this principle of threshold-dependent effector activation. These display regulatory mechanisms paralleling physiological signaling based on 3'-5' cyclic mononucleotides, NAD+-derived messengers, two- and one-component signaling that includes histidine kinase-based signaling, and proteolytic activation. Furthermore, these systems allowed the identification of multiple new sensory signal sensory components, such as a tetratricopeptide repeat (TPR) scaffold predicted to recognize NAD+-derived signals, unreported versions of the STING domain, prokaryotic YEATS domains, and a predicted nucleotide sensor related to receiver domains. We also identify previously unrecognized invader detection components and effector components, such as prokaryotic versions of the Wnt domain. Finally, we show that there have been multiple acquisitions of unidentified STING domains in eukaryotes, while the TPR scaffold was incorporated into the animal immunity/apoptosis signal-regulating kinase (ASK) signalosome.IMPORTANCE Both prokaryotic and eukaryotic immune systems face the dangers of premature activation of effectors and degradation of self-molecules in the absence of an invader. To mitigate this, they have evolved threshold-setting regulatory mechanisms for the triggering of effectors only upon the detection of a sufficiently strong invader signal. This work defines general templates for such regulation in effector-based immune systems. Using this, we identify several previously uncharacterized prokaryotic immune mechanisms that accomplish the regulation of downstream effector deployment by using nucleotide, NAD+-derived, two-component, and one-component signals paralleling physiological homeostasis. This study has also helped identify several previously unknown sensor and effector modules in these systems. Our findings also augment the growing evidence for the emergence of key animal immunity and chromatin regulatory components from prokaryotic progenitors.

Assembly of an atypical \u03b1-macroglobulin complex from Pseudomonas aeruginosa

Alpha-2-macroglobulins (A2Ms) are large spectrum protease inhibitors that are major components of the eukaryotic immune system. Pathogenic and colonizing bacteria, such as the opportunistic pathogen Pseudomonas aeruginosa, also carry structural homologs of eukaryotic A2Ms. Two types of bacterial A2Ms have been identified: Type I, much like the eukaryotic form, displays a conserved thioester that is essential for protease targeting, and Type II, which lacks the thioester and to date has been poorly studied despite its ubiquitous presence in Gram-negatives. Here we show that MagD, the Type II A2M from P. aeruginosa that is expressed within the six-gene mag operon, specifically traps a target protease despite the absence of the thioester motif, comforting its role in protease inhibition. In addition, analytical ultracentrifugation and small angle scattering show that MagD forms higher order complexes with proteins expressed in the same operon (MagA, MagB, and MagF), with MagB playing the key stabilization role. A P. aeruginosa strain lacking magB cannot stably maintain MagD in the bacterial periplasm, engendering complex disruption. This suggests a regulated mechanism of Mag complex formation and stabilization that is potentially common to numerous Gram-negative organisms, and that plays a role in periplasm protection from proteases during infection or colonization.

Evolutionary Analysis of CRISPRs in Archaea: An Evidence for Horizontal Gene Transfer

Akin to the eukaryotic immune system, prokaryotes harbor CRISPRs, a lineup of DNA direct repeats and spacers to foster immunity against the invading phages and plasmids. A CRISPR (Clustered Regularly Interspaced Short Palindrome Repeats) unit found within the genome of an organism consists of an array of repeating sequences interspaced by unique spacers and is associated with special genes that reside adjacent to the array. The spacer sequences are nucleotide fragments integrated from an invading organism. The comparative genomic analysis of CRISPR sequences affirms huge variation within the CRISPR–CAS systems among different prokaryotes. Here, an analysis of the complete archaeal species is directed for their CRISPR sequences along with a case study. The phylogenetic analysis sketched from the CRISPR sequences signifies a harmony along the direct repeats of the analyzed organisms with no trace of spacer similarity. Further, novel CRISPR elements are procured, aside from those formerly present in the database. The CRISPRs are then subjected for local alignment using BLAST to ensure whether any of the sequences showed similarity with the human and viral genomes available in NCBI.

CRISPR–Cas adaptive immunity and developments in CRISPR–Cas applications: Reply to comments on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems”

Available online at www.sciencedirect.com ScienceDirect Physics of Life Reviews 11 (2014) 149–151 www.elsevier.com/locate/plrev Reply to comments CRISPR–Cas adaptive immunity and developments in CRISPR–Cas applications Reply to comments on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems” Edwin L. Cooper ∗ , Nicola Overstreet Laboratory of Comparative Immunology, Department of Neurobiology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095-1763, United States Received 13 December 2013; accepted 17 December 2013 Available online 21 December 2013 Communicated by M. Frank-Kamenetskii We appreciate the commentaries received from Stern [1], Pinti and Cossarizza [2], Plagens and Randau [3], Koonin [4], Wanner et al. [5], and Severinov [6], and their observations and insights, which we will now discuss. As is mentioned in many of the commentaries received, the emergence of knowledge about CRISPR–Cas systems marks an important shift in the mindset of immunology. For a long time, eukaryotic immune systems were viewed through the paradigm of self/not-self. This paradigm was rooted in clonal selection and the dual innate and adaptive model [7–9]. With the advent of the danger hypothesis, it became apparent that the self/not-self pattern was not im- pervious to upheaval [7,10]. Similarly, small RNA systems have shifted the perspective of immunology. When RNA systems were first discovered, they were seen as an exception to the suite of well-known protein-recognition adaptive immune defenses. However, in the words of Pinti and Cossarizza, “it is now clear even to us, human immunologists, that RNA-based defense mechanisms are the rule, and not the exception, and that the modern, adaptive immune system has been built on an ancient basement made by RNA” [2]. Stern similarly remarks that the wealth of RNA systems reveals the “general solutions to the problem of par- asites” [1]. The recognition of not-self motifs, infected cell death, and retained memory of past exposures are all mechanisms of immune defense that are observed across the tree of life [1]. Koonin likens small RNA defense mech- anisms to an idea that evolution found “ ‘too good’ to be abandoned” [4]. We agree that the pervasiveness of small RNA systems in prokaryotes and eukaryotes demonstrates the importance of these systems to life throughout the phy- logenetic tree, and speaks to the power of these types of systems. As Koonin states, “there seems to be underlying logic in their evolution that is both universal and simple” [4]. DOI of original article: http://dx.doi.org/10.1016/j.plrev.2013.11.002. DOIs of comments: http://dx.doi.org/10.1016/j.plrev.2013.11.004, http://dx.doi.org/10.1016/j.plrev.2013.12.001, http://dx.doi.org/10.1016/j.plrev.2013.11.012, http://dx.doi.org/10.1016/j.plrev.2013.12.002, http://dx.doi.org/10.1016/j.plrev.2013.12.003, http://dx.doi.org/10.1016/j.plrev.2013.11.015. * Corresponding author. 1571-0645/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.plrev.2013.12.011

The double-edged sword of Lamarck: Comment on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems” by Edwin L. Cooper and Nicola Overstreet

The double-edged sword of Lamarck: Comment on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems” by Edwin L. Cooper and Nicola Overstreet

What hath DNA wrought? CRISPR-CAS gene silencing and engineering from bacteria to humans: Comment on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems” by Edwin L. Cooper and Nicola Overstreet

What hath DNA wrought? CRISPR-CAS gene silencing and engineering from bacteria to humans: Comment on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems” by Edwin L. Cooper and Nicola Overstreet

CRISPR-Cas: Outstanding questions remain: Comment on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems” by Edwin L. Cooper and Nicola Overstreet

CRISPR-Cas: Outstanding questions remain: Comment on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems” by Edwin L. Cooper and Nicola Overstreet

Small RNA-guided adaptive immunity: Comment on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems” by Cooper and Overstreet

Small RNA-guided adaptive immunity: Comment on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems” by Cooper and Overstreet

Small RNAs in prokaryotes and eukaryotes: A lesson for human immunologists: Comment on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems” by Edwin L. Cooper and Nicola Overstreet

The manipulation of the immune system by artificially induced, highly specific responses to what we name “antigens” has been a common medical practice for more than 4000 years since in ancient China it became a common practice to provoke a mild form of variola major to protect individuals from the more dangerous natural infection. When we think of immunity and immune system, the most obvious image that comes to our mind is related to the use of vaccines, and thus to the complex responses started by the adaptive immune system of vertebrates. Accordingly, the immune system of humans – and for some researchers also that of mice – is usually considered “the” immune system. The paper by Cooper and Overstreet [1] carefully reviews the CRISPR–Cas system in the prokaryotes, and the miRNAs, siRNAs and piRNAs systems in the eukaryotes, recapitulating their mechanisms of action and their possible convergent or divergent evolution. Cooper and Overstreet challenge in several aspects the common way of thinking about the concept of immunology that is common to many researchers, including us, who focused on the vertebrate immune system. First, the wide look on small RNAs as defence system clearly points out that the innate and adaptive immunity of vertebrates represents a striking evolutionary exception, rather than the general rule. Furthermore, the presence of such a huge number of piRNAs in our genome suggest that, probably, many aspects of RNA-based defences in vertebrates, including humans, have still to be explored, especially taking into account the large knowledge existing in the field of adaptive immunity. A deeper analysis of miRNAs and piRNAs in humans – that is at its dawn, as Cooper and Overstreet suggest – will be extremely helpful for finding new, unexplored tools for controlling not only selfish genetic elements present in our genome, but hopefully to better understand the biology and the response to viruses or retroviruses that currently we are unable to control. Indeed, several viruses such as the human immunodeficiency

CRISPR and RNA interference: similarities across immune systems: Comment on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems” by Edwin L. Cooper and Nicola Overstreet

CRISPR and RNA interference: similarities across immune systems: Comment on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems” by Edwin L. Cooper and Nicola Overstreet

CRISPR-Mediated Adaptive Immune Systems in Bacteria and Archaea

Effective clearance of an infection requires that the immune system rapidly detects and neutralizes invading parasites while strictly avoiding self-antigens that would result in autoimmunity. The cellular machinery and complex signaling pathways that coordinate an effective immune response have generally been considered properties of the eukaryotic immune system. However, a surprisingly sophisticated adaptive immune system that relies on small RNAs for sequence-specific targeting of foreign nucleic acids was recently discovered in bacteria and archaea. Molecular vaccination in prokaryotes is achieved by integrating short fragments of foreign nucleic acids into a repetitive locus in the host chromosome known as a CRISPR (clustered regularly interspaced short palindromic repeat). Here we review the mechanisms of CRISPR-mediated immunity and discuss the ecological and evolutionary implications of these adaptive defense systems.

Biochemical, Structural and Molecular Dynamics Analyses of the Potential Virulence Factor RipA from Yersinia pestis

Human diseases are attributed in part to the ability of pathogens to evade the eukaryotic immune systems. A subset of these pathogens has developed mechanisms to survive in human macrophages. Yersinia pestis, the causative agent of the bubonic plague, is a predominately extracellular pathogen with the ability to survive and replicate intracellularly. A previous study has shown that a novel rip (required for intracellular proliferation) operon (ripA, ripB and ripC) is essential for replication and survival of Y. pestis in postactivated macrophages, by playing a role in lowering macrophage-produced nitric oxide (NO) levels. A bioinformatics analysis indicates that the rip operon is conserved among a distally related subset of macrophage-residing pathogens, including Burkholderia and Salmonella species, and suggests that this previously uncharacterized pathway is also required for intracellular survival of these pathogens. The focus of this study is ripA, which encodes for a protein highly homologous to 4-hydroxybutyrate-CoA transferase; however, biochemical analysis suggests that RipA functions as a butyryl-CoA transferase. The 1.9 Å X-ray crystal structure reveals that RipA belongs to the class of Family I CoA transferases and exhibits a unique tetrameric state. Molecular dynamics simulations are consistent with RipA tetramer formation and suggest a possible gating mechanism for CoA binding mediated by Val227. Together, our structural characterization and molecular dynamic simulations offer insights into acyl-CoA specificity within the active site binding pocket, and support biochemical results that RipA is a butyryl-CoA transferase. We hypothesize that the end product of the rip operon is butyrate, a known anti-inflammatory, which has been shown to lower NO levels in macrophages. Thus, the results of this molecular study of Y. pestis RipA provide a structural platform for rational inhibitor design, which may lead to a greater understanding of the role of RipA in this unique virulence pathway.