key: cord-0003277-fqdv3ohv authors: Arroyo-Olarte, Ruben Dario; Thurow, Laura; Kozjak-Pavlovic, Vera; Gupta, Nishith title: Illuminating pathogen–host intimacy through optogenetics date: 2018-07-12 journal: PLoS Pathog DOI: 10.1371/journal.ppat.1007046 sha: 7f67a850db06d9b9e3f63d9943e2b1fc5a72ca8f doc_id: 3277 cord_uid: fqdv3ohv The birth and subsequent evolution of optogenetics has resulted in an unprecedented advancement in our understanding of the brain. Its outstanding success does usher wider applications; however, the tool remains still largely relegated to neuroscience. Here, we introduce selected aspects of optogenetics with potential applications in infection biology that will not only answer long-standing questions about intracellular pathogens (parasites, bacteria, viruses) but also broaden the dimension of current research in entwined models. In this essay, we illustrate how a judicious integration of optogenetics with routine methods can illuminate the host–pathogen interactions in a way that has not been feasible otherwise. and/or by using inducible expression systems. The advantages of optogenetics so far have far outweighed the stated concerns, as evident by its exceptional success. Originally, optogenetics was comprised of light-activated proteins that can modify membrane potential or allow control of signal cascades, molecular interactions, and gene expression [2, 3] . The ever-expanding field now, in its broadest sense, includes gene-encoded light-sensitive sensors, which can be deployed to gauge intracellular messengers or metabolites. Not least, the method has also embraced technological procedures to deliver light-regulated proteins, to control the illumination, and to measure the outcome [1] [2] [3] . Currently, more than 40 types of optogenetic actuators and around 30 biosensors are available according to Addgene repository (www.addgene.org). It is worth noting that many of them have been catered to address a wide range of hypotheses in neurobiology. While other research fields have begun adopting them to meet specific objectives, such as to study the stage differentiation in intracellular parasites [4] or to fine-tune the function of immune cells [5] , applications of light-activated proteins remain extremely limited. We believe that the technique is now well primed to answer prevailing questions and shepherd new avenues in infection research. Herein, we outline comprehensive applications of optogenetics to study various paradigms embracing intracellular parasites, bacteria, and viruses of clinical as well as veterinary relevance (Table 1 and Fig 1) . Specifically, Fig 1A shows selected opto-tools to modulate or sense secondary messengers (cyclic nucleotides) and physicochemical parameters (pH, reactive oxygen species [ROS] , ions), whereas Fig 1B highlights the light-induced control of gene editing, protein expression, and lipidic signaling. A list of relevant references including Addgene construct numbers is included as supporting information (S1 Appendix). The text below explains how best we can deploy them to examine notable events during asexual reproduction of archetypical pathogens (Fig 2) . Although we outline only designated pairs of optogenetic proteins and pathogens, in principle, most systems are equally applicable to all genetically tractable organisms. In fact, with the advent of CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) and related genome engineering tools in common parasitic protists [6] and other microorganisms [7] , it has become easier than ever to make desired optogenetic strains in "model" as well as "nonmodel" pathogens. Parasites include an assorted group of eukaryotic pathogens taking advantage of the host, which itself is also a eukaryotic organism. The study of the complex relationship between parasites and host cells is often compromised because chemical modulators usually cannot distinguish between targets conserved in both entities. Likewise, a spatiotemporal detection of universal metabolites in intracellularly residing parasites is simply not possible via chemical methods. Besides troubleshooting these issues, making of optogenetic strains allows us to literally tell the pathogen (or host cell) when and where to modulate or monitor the cascade and for how long and how much. A pioneering study involving expression of a light-activated adenylate cyclase in the protozoan parasite Toxoplasma gondii has already demonstrated the proof of principle for applying optogenetics in infection research [4] . Equally, expression of gene-encoded biosensors has enabled an otherwise challenging monitoring of subcellular calcium in T. gondii and Plasmodium falciparum enclosed within host cells [8, 9] . These works have indeed paved a way to tap the vast potential of optogenetic actuators and biosensors. One can, for example, evaluate the roles of specific molecules during various parasitic stages, as epitomized by T. gondii and Trypanosoma cruzi (Fig 2A) . Some of the most fascinating applications in parasites involve perturbation of ion homeostasis by light-gated channels, as well as photo-oscillation of calcium, cNMP (cyclic nucleotide monophosphate), and phosphoinositide signaling cascades. In essence, a rigorous experimental design can permit systematic dissection of signaling by studying optically induced changes in histone coding, transcriptome, proteome, and metabolome. A repertoire of cation-and anion-specific channelrhodopsin variants is available to investigate inter-or intra-organelle ion homeostasis [10] . Evenly, genetically encoded calcium actuators (GECAs) [11, 12] and light-induced cyclic nucleotide cyclases [13] [14] [15] [16] , along with corresponding phosphodiesterases [17, 18] , are perfectly poised to elucidate novel aspects of calcium and cNMP signaling, respectively. For instance, the parasite strains expressing a light-activated adenylate or guanylate cyclase in a PKA-(cAMP-dependent protein kinase) or PKG-deficient (cGMP-dependent protein kinase) mutant can be subjected to phosphoproteomic analysis to identify the core signaling mediators. On a different note, the method may even prove beneficial over ablation of native signaling proteins in certain cases because a sophisticated reversible control can be achieved as opposed to all or none effect in the gene-knockout mutants. This is well exemplified by applying optogenetics in T. gondii [4] , in which induction of parasite-derived cytosolic cAMP can exert contrary effects depending on the duration and intensity of the stimulus. In this regard, it does make sense to couple optical regulation with a real-time detection using apposite sensors. A gamut of color-tuned biosensors, such as for calcium [19] [20] [21] , cAMP [22] , cGMP [23] , c-di-GMP [24] , DAG (diacylglycerol) [25] , and IP 3 [26] , is available to quantify subcellular oscillations. Conversely, the above tools can be expressed in host cells to study the influence of host milieu on parasites. One such example is to dissect the mechanism of action of antitrypanocidal drugs, which control the muscle function by apparent modulation of calcium homeostasis during chronic Chagas disease [27] . These drugs are also active against Leishmania [28] , further advocating the utility of calcium releasers and sensors (Table 1) . Other appealing optogenetic approaches involve engineering host cells or parasites to harbor reactive oxygen species generating proteins (RGPs) [29] , as well as the biosensors of lipids [25, 30, 31] , polar metabolites [32] [33] [34] , nitric oxide [35] , voltage [36] , redox [37, 38] , and pH [39] [40] [41] , each of them tailored to address specific paradigms and individual needs (Table 1) . Just as with intracellular parasites, infection with prokaryotic pathogens can be examined from the side of the bacteria as well as from the host-cell side. Yet again, studying pathogen-host interactions has so far not relied on the deployment of optogenetics. Table 1 lists some customary optogenetic tools, which are projected to elucidate the mechanisms underlying the uptake of bacteria, survival within host cells, or transcytosis for leading human pathogens including but not limited to Mycobacteria, Chlamydia, Salmonella, and Staphylococcus. As depicted in Fig 1A and [13, 14] , cGMP [15, 16] , or c-di-GMP [42, 43] . It is also possible to develop light-responsive expression systems using photocaged effectors, such as doxycycline [44] and IPTG (isopropyl β-D-1-thiogalactopyranoside) [45] or proteins [46, 47] , which are released or activated upon illumination to control the gene expression or protein activity. Just recently, the LOV2-ODC-degron system has been reported, which targets the conjugated protein of interest to light-dependent proteasomal degradation, thereby controlling the protein stability [48] (Fig 1B) . Even though most of these tools have been originally developed in eukaryotic cells, their tailored adoption in prokaryotes is quite plausible. Other comparable applications include modulation of protein-protein interactions, which could in turn be used to regulate cell signaling, genome editing, endogenous transcription, and epigenetic states [49] [50] [51] [52] [53] (Fig 1B) . Equally, gene-encoded sensors can be applied to monitor the pH, ions, membrane potential, redox states, temperature, pressure, and molecular crowding [54] . Similar approaches can be used to modify host cells and regulate gene expression, signaling, autophagy, and organelle functions-processes that are considered important for interactions of host cells with bacterial pathogens, e.g., Chlamydia (Fig 2A) . Optogenetics also enables the control of organelle transport and positioning [55] , which might be useful when studying the importance of host organelle hijacking by microbes. It is even possible to induce and repress the mechanotransduction and cellular forces with spatiotemporal accuracy [56] . The underlying method involves controlling the subcellular activation of RhoA using the CRY2/CIBN light-gated dimerizer system [50] . In order to apply these methods, some hurdles need to be overcome, especially when manipulating mammalian host cells. The first one is the delivery of optogenetic proteins, which can be best solved by making stable transgenic lines using the established viral expression systems. Another issue is the compartmentalization, i.e., the targeting of a tool to the chosen organelle, such as nucleus, endoplasmic reticulum, and mitochondria. It can be resolved, though, by introducing organelle-specific signal sequences into the protein of interest. Parallel measurement of metabolites and ions in host cells harboring a pathogen is another bottleneck, which can be solved by coexpression of suitable biosensors. Once inducted, an effective commissioning of these tools is expected to shed light on numerous processes that have remained shadowy for a while. biosensors of lipids and lipid-derived metabolites. Upon illumination, an RNA-guided dCas9 binds to a CRY2-VP64 transactivation domain, which in turn allows otherwise repressed transcription of a gene. LoxP-mediated recombination at a target locus is achieved by a photo-dimerizable CRE recombinase. Light-activated degron: The protein of interest is fused to a photosensitive LOV2 and a proteasome targeting cODC1 domain. Optically induced degradation is facilitated by a conformational shift in the latter 2 domains. CRY2/CIBN fusion to inositol phosphatase enables a concurrent modulation and evaluation of phosphoinositide metabolism. Lipid-binding domains Lact-C2 and PKCδ-C1 fused to GFP allow fluorescent detection of subcellular PtdSer and DAG, respectively. Equally, a fusion of CFP and Venus with IP 3 -binding motif permits a FRET-based monitoring of IP 3 . Further details on indicated proteins can be found in S1 Appendix and references therein. CFP, cyan-fluorescent protein; CIBN, N-terminus of CIB1; CRE, cyclization recombinase; DAG, diacylglycerol; FRET, fluorescence-resonance energy transfer; GFP, green fluorescent protein; LACE, light-activated CRISPR-Cas9 effector. https://doi.org/10.1371/journal.ppat.1007046.g001 Viruses are master modulators of signaling, immune response, and metabolism in the infected host [57] [58] [59] . In a way similar to eukaryotic and prokaryotic pathogens, most applications are also applicable to viruses (Table 1) ; although, optogenetic tools have to be targeted primarily to the host cell ( Fig 2B) . For example, opsin and GECA family proteins allow in-depth examination of the role of ions in promoting or demoting the viral lifecycle. One could envisage optogenetically eliciting a release of Ca 2+ from the endoplasmic reticulum (naturally occurring via IP 3 R in HIV-1, HSV [herpes simplex virus], rotavirus), activation of PLC pathway (mediated via GPCRs in rotavirus), impairment of SERCA (sarco-endoplasmic reticulum calcium ATPase), and control of membrane permeability (via viroporin in HIV-1, HCV, influenza virus, coronavirus) through light-gated channels or pumps [60, 61] . Phosphoinositide actuators [62] can be applied to control early steps of attachment or fusion to the host membrane in the case of Ebola virus, coronavirus, and HIV-1 [63] [64] [65] . Other potential usages include lightmediated regulation of immune response (e.g., activation of HIV-infected CD4 cells), killing of virus-infected host cells (e.g., IFN Y in CD8 + and NK cells), and photo-editing of viral genomes. Similarly, ROS-yielding proteins, physicochemical actuators, protein recruiters/oligomerizers, nucleotide cyclases, and lipid-derived mediators allow us to study other enigmatic aspects ( Table 1) . As indicated above, viruses can also be used as vectors for delivering optogenetic tools into specific cell populations or tissues in mammalian cells. Addgene repository contains several lentiviral constructs for targeted delivery and integration into genomes. Additional popular models include adeno-associated virus [66] and rabies virus [67] . Not least, selected geneencoded sensors that can facilitate optogenetic work in virology include GECIs (gene-encoded calcium indicators), cNMP biosensors, as well as fluorescent indicators for pH, lipids, and several other metabolites (Table 1) . Among many, one assay would be to test the pH dependence of membrane fusion during the entry of influenza and stomatitis viruses, mediated by hemagglutinin and G glycoprotein, respectively [68, 69] . Another conceivable application is to visualize lipid rafts as a predicted platform for the entry, assembly, and release of viral particles [70] [71] [72] . Finally, a fusion approach coutilizing optical actuators and sensors will certainly embolden existing toolboxes in virology. A modulation and monitoring of pathogen-specific pathways without affecting the sheltering host cell is nearly impossible with chemical modulators and fluorophores. Contrariwise, selective manipulation of the infected host is equally challenging. Although not common yet, . Note that only selected features are highlighted. The shared events include invasion, proliferation, and egress. The tachyzoite stage of T. gondii actively invades host cells, reorders several organelles (not depicted for simplicity), replicates by endodyogeny in a nonfusogenic vacuole, and then exits by lysing the vacuolar and host membranes. Cyclic nucleotides (cAMP, cGMP) and ions (Ca 2+ , K + ) play very important roles during the lytic cycle. The trypomastigote stage of T. cruzi enters the host cell by recruiting lysosomes and then escapes into cytoplasm (mediated by TcTox), where they reproduce asexually as amastigotes. Among others, Ca 2+ , pH, and ROS are major factors during T. cruzi infection. The EBs of Chlamydia are endocytosed into membranous vacuoles, which fuse to form an inclusion, the replicative compartment. Later on, they differentiate into larger metabolically active RBs, which replicate by binary fission before converting back to EBs. Similar to tachyzoites, Chlamydia is known to intercept/recruit many host organelles, such as Golgi, lipid droplets, and endolysosomes, probably for acquiring nutrients. Again, cAMP and cGMP, along with prokaryote-specific c-di-GMP, control the stage differentiation and STING-mediated modulation of host immunity genes, respectively. (B) Abridged lifecycle of viruses infecting a host cell. Key second messengers, ions, and metabolites potentially regulatable or detectable by optogenetic means are shown in relation to specific events during the course of infection. In particular, calcium, pH, ROS, and phosphoinositide signaling regulate a repertoire of phenomena. For additional details, please refer to the table outlining different tools, pathogens, and paradigms ( optogenetics offers an imperative toolbox to modify and monitor subcellular processes both in the host as well as in the pathogen. The deployment of optogenetics shall overcome most, if not all, difficulties inherent to classic approaches in infection research. Just as with neurosciences, this will hopefully also lead to improvement of old tools and discovery of customized solutions catering pathogens in imminent future. Hence, building a bridge between the fields of optogenetics and infection biology remains more important than ever. Supporting information S1 Appendix. Major optogenetic tools and underlying source. (PDF) Illuminating cell signalling with optogenetic tools Optogenetics: controlling cell function with light Optogenetic modulation of an adenylate cyclase in Toxoplasma gondii demonstrates a requirement of the parasite cAMP for host-cell invasion and stage differentiation Near-infrared photoactivatable control of Ca 2+ signaling and optogenetic immunomodulation Taming parasites by tailoring them CRISPR-based genomic tools for the manipulation of genetically intractable microorganisms Optogenetic monitoring identifies phosphatidylthreonine-regulated calcium homeostasis in Toxoplasma gondii Ca 2+ monitoring in Plasmodium falciparum using the yellow Cameleon-Nano biosensor The form and function of channelrhodopsin Optogenetic toolkit for precise control of calcium signaling Optogenetic control of endogenous Ca 2+ channels in vivo Natural and engineered photoactivated nucleotidyl cyclases for optogenetic applications Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa A rhodopsin-guanylyl cyclase gene fusion functions in visual perception in a fungus Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp Structure and mechanism of a bacterial light-regulated cyclic nucleotide phosphodiesterase Engineering of a red-light-activated human cAMP/cGMP-specific phosphodiesterase Spontaneous network activity visualized by ultrasensitive Ca 2+ indicators, yellow Cameleon-Nano An expanded palette of genetically encoded Ca 2+ indicators Ultrasensitive fluorescent proteins for imaging neuronal activity Interrogating cyclic AMP signaling using optical approaches Fluorescent sensors for rapid monitoring of intracellular cGMP Next-generation RNA-based fluorescent biosensors enable anaerobic detection of cyclic di-GMP Imaging diacylglycerol dynamics at organelle membranes Use of fluorescence resonance energy transfer-based biosensors for the quantitative analysis of inositol 1,4,5-trisphosphate dynamics in calcium oscillations The emerging role of amiodarone and dronedarone in Chagas disease Amiodarone destabilizes intracellular Ca 2+ homeostasis and biosynthesis of sterols in Leishmania mexicana Optogenetic control of ROS production Membrane phosphatidylserine regulates surface charge and protein localization Genetically encoded probes for phosphatidic acid Fluorescence imaging of cellular metabolites with RNA Synthetic biosensors for precise gene control and real-time monitoring of metabolites Genetically encoded sensors enable real-time observation of metabolite production Imaging the nanomolar range of nitric oxide with an amplifier-coupled fluorescent indicator in living cells Voltage imaging with genetically encoded indicators Imaging cytosolic NADH-NAD + redox state with a genetically encoded fluorescent biosensor Monitoring thioredoxin redox with a genetically encoded red fluorescent biosensor Visualizing secretion and synaptic transmission with pHsensitive green fluorescent proteins Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins SRpHi ratiometric pH biosensors for super-resolution microscopy Optogenetic module for dichromatic control of c-di-GMP signaling Using light-activated enzymes for modulating intracellular c-di-GMP levels in bacteria Doxycyclinedependent photoactivated gene expression in eukaryotic systems Light-induced gene expression with photocaged IPTG for induction profiling in a high-throughput screening system From dusk till dawn: one-plasmid systems for light-regulated gene expression Blue light-mediated transcriptional activation and repression of gene expression in bacteria Development of a synthetic switch to control protein stability in eukaryotic cells with light Spatiotemporal control of cell signalling using a lightswitchable protein interaction Rapid blue-light-mediated induction of protein interactions in living cells Optical control of mammalian endogenous transcription and epigenetic states A light-inducible CRISPR-Cas9 system for control of endogenous gene activation Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase Design and development of genetically encoded fluorescent sensors to monitor intracellular chemical and physical parameters Optogenetic control of organelle transport and positioning Optogenetic control of cellular forces and mechanotransduction Subversion of cell signaling by pathogens Viral manipulation of the host immune response Stealing the keys to the kitchen: Viral manipulation of the host cell metabolic network Viral calciomics: Interplays between Ca 2+ and virus Relevance of viroporin ion channel activity on viral replication and pathogenesis Optogenetic control of phosphoinositide metabolism Phosphoinositide-3 kinase-Akt pathway controls cellular entry of Ebola virus Phosphatidylinositol 4-kinase IIIβ is required for severe acute respiratory syndrome coronavirus spike-mediated cell entry PIP2: choreographer of actin-adaptor proteins in the HIV-1 dance Virally mediated optogenetic excitation and inhibition of pain in freely moving nontransgenic mice Rabies virus CVS-N2cΔG strain enhances retrograde synaptic transfer and neuronal viability Structure of influenza haemagglutinin at the pH of membrane fusion Mechanism of membrane fusion induced by vesicular stomatitis virus G protein Plasma membrane rafts play a critical role in HIV-1 assembly and release Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its colocalization with CD163 in lipid rafts Dynamin-and lipid raft-dependent entry of decay-accelerating factor (DAF)-binding and non-DAF-binding Coxsackieviruses into nonpolarized Cells We thank Dr. Bertha Espinoza for comments on the Trypanosoma cruzi lifecycle.