Potent Activity of Hybrid Arthropod Antimicrobial Peptides Linked by Glycine Spacers.

Arthropod antimicrobial peptides (AMPs) offer a promising source of new leads to address the declining number of novel antibiotics and the increasing prevalence of multidrug-resistant bacterial pathogens. AMPs with potent activity against Gram-negative bacteria and distinct modes of action have been identified in insects and scorpions, allowing the discovery of AMP combinations with additive and/or synergistic effects. Here, we tested the synergistic activity of two AMPs, from the dung beetle Copris tripartitus (CopA3) and the scorpion Heterometrus petersii (Hp1090), against two strains of Escherichia coli. We also tested the antibacterial activity of two hybrid peptides generated by joining CopA3 and Hp1090 with linkers comprising two (InSco2) or six (InSco6) glycine residues. We found that CopA3 and Hp1090 acted synergistically against both bacterial strains, and the hybrid peptide InSco2 showed more potent bactericidal activity than the parental AMPs or InSco6. Molecular dynamics simulations revealed that the short linker stabilizes an N-terminal 310-helix in the hybrid peptide InSco2. This secondary structure forms from a coil region that interacts with phosphatidylethanolamine in the membrane bilayer model. The highest concentration of the hybrid peptides used in this study was associated with stronger hemolytic activity than equivalent concentrations of the parental AMPs. As observed for CopA3, the increasing concentration of InSco2 was also cytotoxic to BHK-21 cells. We conclude that AMP hybrids linked by glycine spacers display potent antibacterial activity and that the cytotoxic activity can be modulated by adjusting the nature of the linker peptide, thus offering a strategy to produce hybrid peptides as safe replacements or adjuncts for conventional antibiotic therapy.

An E460D Substitution in the NS5 Protein of Tick-Borne Encephalitis Virus Confers Resistance to the Inhibitor Galidesivir (BCX4430) and Also Attenuates the Virus for Mice.

The adenosine analogue galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under clinical development for treatment of Ebola and yellow fever virus infections. Moreover, galidesivir also inhibits the reproduction of tick-borne encephalitis virus (TBEV) and numerous other medically important flaviviruses. Until now, studies of this antiviral agent have not yielded resistant viruses. Here, we demonstrate that an E460D substitution in the active site of TBEV RNA-dependent RNA polymerase (RdRp) confers resistance to galidesivir in cell culture. Galidesivir-resistant TBEV exhibited no cross-resistance to structurally different antiviral nucleoside analogues, such as 7-deaza-2'-C-methyladenosine, 2'-C-methyladenosine, and 4'-azido-aracytidine. Although the E460D substitution led to only a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in vivo, with a 100% survival rate and no clinical signs observed in infected mice. Furthermore, no virus was detected in the sera, spleen, or brain of mice inoculated with the galidesivir-resistant TBEV. Our results contribute to understanding the molecular basis of galidesivir antiviral activity, flavivirus resistance to nucleoside inhibitors, and the potential contribution of viral RdRp to flavivirus neurovirulence.IMPORTANCE Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in Europe and Asia and for which there is currently no specific therapy. We have previously found that galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, which is under clinical development for treatment of Ebola and yellow fever virus infections, has a strong antiviral effect against TBEV. For any antiviral drug, it is important to generate drug-resistant mutants to understand how the drug works. Here, we produced TBEV mutants resistant to galidesivir and found that the resistance is caused by a single amino acid substitution in an active site of the viral RNA-dependent RNA polymerase, an enzyme which is crucial for replication of the viral RNA genome. Although this substitution led only to a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in a mouse model. Our results contribute to understanding the molecular basis of galidesivir antiviral activity.

Escape of Tick-Borne Flavivirus from 2'-C-Methylated Nucleoside Antivirals Is Mediated by a Single Conservative Mutation in NS5 That Has a Dramatic Effect on Viral Fitness.

Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in humans. Despite its high medical relevance, no specific antiviral therapy is currently available. Here we demonstrate that treatment with a nucleoside analog, 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), substantially improved disease outcomes, increased survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2'-CMA, two drug-resistant TBEV clones were generated and characterized. The two clones shared a signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This mutation conferred resistance to various 2'-C-methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'-C-azidocytidine and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187). All-atom molecular dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that coordinates the position of a metal ion cofactor as 2'-C-methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV strain (Oshima-IC) generated from an infectious cDNA clone and into a TBEV replicon that expresses a reporter luciferase gene (Oshima-REP-luc2A). The mutants were replication impaired, showing reduced growth and a small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These results indicate that TBEV resistance to 2'-C-methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of the viral NS5 RdRp and is associated with strong attenuation of the virus.IMPORTANCE This study found that the nucleoside analog 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-deaza-2'-CMA resulted in significantly higher survival rates and reduced the severity of neurological signs of the disease. Thus, this compound shows promise for further development as an anti-TBEV drug. It is important to generate drug-resistant mutants to understand how the drug works and to develop guidelines for patient treatment. We generated TBEV mutants that were resistant not only to 7-deaza-2'-CMA but also to a broad range of other 2'-C-methylated antiviral medications. Our findings suggest that combination therapy may be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.

Flaviviridae viruses use a common molecular mechanism to escape nucleoside analogue inhibitors.

The RNA-dependent RNA polymerases of Flaviviridae viruses are crucial for replication. The Flaviviridae polymerase is organized into structural motifs (A-G), with motifs F, A, C and E containing interrogating, priming and catalytic substrate-interacting sites. Modified nucleoside analogues act as antiviral drugs by targeting Flaviviridae polymerases and integrating into the synthesized product causing premature termination. A threonine mutation of a conserved serine residue in motif B of Flaviviridae polymerases renders resistance to 2'-C-methylated nucleoside analogues. The mechanism how this single mutation causes Flaviviridae viruses to escape nucleoside analogues is not yet known. Given the pivotal position of the serine residue in motif B that supports motif F, we hypothesized the threonine mutation causes alterations in nucleoside exploration within the entry tunnel. Implementing a stochastic molecular software showed the all-atom 2'-C-methylated analogue reaction within the active sites of wild type and serine-threonine mutant polymerases from Hepacivirus and Flavivirus. Compared with the wild type, the serine-threonine mutant polymerases caused a significant decrease of analogue contacts with conserved interrogating residues in motif F and a displacement of metal ion cofactors. The simulations significantly showed that during the analogue exploration of the active site the hydrophobic methyl group in the serine-threonine mutant repels water-mediated hydrogen bonds with the 2'-C-methylated analogue, causing a concentration of water-mediated bonds at the substrate-interacting sites. Collectively, the data are an insight into a molecular escape mechanism by Flaviviridae viruses from 2'-C-methylated nucleoside analogue inhibitors.

An all-atom, active site exploration of antiviral drugs that target Flaviviridae polymerases.

Natural 2'-modified nucleosides are the most widely used antiviral therapy. In their triphosphorylated form, also known as nucleotide analogues, they target the active site of viral polymerases. Viral polymerases have an overall right-handed structure that includes the palm, fingers and thumb domains. These domains are further subdivided into structurally conserved motifs A-G, common to all viral polymerases. The structural motifs encapsulate the allosteric/initiation (N1) and orthosteric/catalytic (N2) nucleotide-binding sites. The current study investigated how nucleotide analogues explore the N2 site of viral polymerases from three genera of the family Flaviviridae using a stochastic, biophysical, Metropolis Monte Carlo-based software. The biophysical simulations showed a statistical distinction in nucleotide-binding energy and exploration between phylogenetically related viral polymerases. This distinction is clearly demonstrated by the respective analogue contacts made with conserved viral polymerase residues, the heterogeneous dynamics of structural motifs, and the orientation of the nucleotide analogues within the N2 site. Being able to simulate what occurs within viral-polymerase-binding sites can prove useful in rational drug designs against viruses.

Psychological hardiness predicts cardiovascular health.

Many, but not all people experience diminished health, performance and well-being as a function of exposure to stress. However, the underlying neurophysiological processes which characterize hardy or resilient people are not well understood. This study examines psychological hardiness and several indicators of cardiovascular health, including body mass index (BMI) and blood cholesterol markers in a sample of 338 middle-aged adults enrolled in a national security education program. Hierarchical regression analyses reveal that after controlling for the influence of age and sex, high hardiness is related to higher HDL - high density lipoprotein and less body fat (BMI). Lower hardiness is associated with greater total cholesterol to HDL ratio, a cardiovascular disease risk factor. These results suggest that psychological hardiness confers resilience in part through an influence on cholesterol production and metabolism.

Fast evolutionary rates associated with functional loss in class I glucose transporters of Schistosoma mansoni.

BACKGROUND: The trematode parasite, Schistosoma mansoni, has evolved to switch from oxidative phosphorylation to glycolysis in the presence of glucose immediately after invading the human host. This metabolic switch is dependent on extracellular glucose concentration. Four glucose transporters are encoded in the genome of S. mansoni, however, only two were shown to facilitate glucose diffusion. RESULTS: By modeling the phase of human host infection, we showed that transporter transcript expression profiles of recently transformed schistosomula have two opposing responses to increased glucose concentrations. Concurring with the transcription profiles, our phylogenetic analyses revealed that S. mansoni glucose transporters belong to two separate clusters, one associated with class I glucose transporters from vertebrates and insects, and the other specific to parasitic Platyhelminthes. To study the evolutionary paths of both groups and their functional implications, we determined evolutionary rates, relative divergence times, genomic organization and performed structural analyses with the protein sequences. We finally used the modelled structures of the S. mansoni glucose transporters to biophysically (i) analyze the dynamics of key residues during glucose binding, (ii) test glucose stability within the active site, and (iii) demonstrate glucose diffusion. The two S. mansoni Platyhelminthes-specific glucose transporters, which seem to be younger than the other two, exhibit slower rates of molecular evolution, are encoded by intron-poor genes, and transport glucose. Interestingly, our molecular dynamic analyses suggest that S. mansoni class I glucose transporters are not able to transport glucose. CONCLUSIONS: The glucose transporter family in S. mansoni exhibit different evolutionary histories. Our results suggested that S. mansoni class I glucose transporters lost their capacity to transport glucose and that this function evolved independently in the Platyhelminthes-specific glucose transporters. Finally, taking into account the differences in the dynamics of glucose transport of the Platyhelminthes-specific transporters of S. mansoni compared to that of humans, we conclude that S. mansoni glucose transporters may be targets for rationally designed drugs against schistosomiasis.

Nucleoside inhibitors of tick-borne encephalitis virus.

Tick-borne encephalitis virus (TBEV) is a leading cause of human neuroinfections in Europe and Northeast Asia. There are no antiviral therapies for treating TBEV infection. A series of nucleoside analogues was tested for the ability to inhibit the replication of TBEV in porcine kidney cells and human neuroblastoma cells. The interactions of three nucleoside analogues with viral polymerase were simulated using advanced computational methods. The nucleoside analogues 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), 2'-C-methyladenosine (2'-CMA), and 2'-C-methylcytidine (2'-CMC) inhibited TBEV replication. These compounds showed dose-dependent inhibition of TBEV-induced cytopathic effects, TBEV replication (50% effective concentrations [EC50]of 5.1 ± 0.4 µM for 7-deaza-2'-CMA, 7.1 ± 1.2 µM for 2'-CMA, and 14.2 ± 1.9 µM for 2'-CMC) and viral antigen production. Notably, 2'-CMC was relatively cytotoxic to porcine kidney cells (50% cytotoxic concentration [CC50] of ∼50 µM). The anti-TBEV effect of 2'-CMA in cell culture diminished gradually after day 3 posttreatment. 7-Deaza-2'-CMA showed no detectable cellular toxicity (CC50 > 50 µM), and the antiviral effect in culture was stable for >6 days posttreatment. Computational molecular analyses revealed that compared to the other two compounds, 7-deaza-2'-CMA formed a large cluster near the active site of the TBEV polymerase. High antiviral activity and low cytotoxicity suggest that 7-deaza-2'-CMA is a promising candidate for further investigation as a potential therapeutic agent in treating TBEV infection.

Tribolium castaneum defensins are primarily active against Gram-positive bacteria.

The red flour beetle Tribolium castaneum is a destructive insect pest of stored food and feed products, and a model organism for development, evolutionary biology and immunity. The insect innate immune system includes antimicrobial peptides (AMPs) with a wide spectrum of targets including viruses, bacteria, fungi and parasites. Defensins are an evolutionarily-conserved class of AMPs and a potential new source of antimicrobial agents. In this context, we report the antimicrobial activity, phylogenetic and structural properties of three T. castaneum defensins (Def1, Def2 and Def3) and their relevance in the immunity of T. castaneum against bacterial pathogens. All three recombinant defensins showed bactericidal activity against Micrococcus luteus and Bacillus thuringiensis serovar tolworthi, but only Def1 and Def2 showed a bacteriostatic effect against Staphylococcus epidermidis. None of the defensins showed activity against the Gram-negative bacteria Escherichia coli and Pseudomonas entomophila or against the yeast Saccharomyces cerevisiae. All three defensins were transcriptionally upregulated following a bacterial challenge, suggesting a key role in the immunity of T. castaneum against bacterial pathogens. Phylogenetic analysis showed that defensins from T. castaneum, mealworms, Udo longhorn beetle and houseflies cluster within a well-defined clade of insect defensins. We conclude that T. castaneum defensins are primarily active against Gram-positive bacteria and that other AMPs may play a more prominent role against Gram-negative species.

Understanding the evolutionary structural variability and target specificity of tick salivary Kunitz peptides using next generation transcriptome data.

BACKGROUND: Ticks are blood-sucking arthropods and a primary function of tick salivary proteins is to counteract the host's immune response. Tick salivary Kunitz-domain proteins perform multiple functions within the feeding lesion and have been classified as venoms; thereby, constituting them as one of the important elements in the arms race with the host. The two main mechanisms advocated to explain the functional heterogeneity of tick salivary Kunitz-domain proteins are gene sharing and gene duplication. Both do not, however, elucidate the evolution of the Kunitz family in ticks from a structural dynamic point of view. The Red Queen hypothesis offers a fruitful theoretical framework to give a dynamic explanation for host-parasite interactions. Using the recent salivary gland Ixodes ricinus transcriptome we analyze, for the first time, single Kunitz-domain encoding transcripts by means of computational, structural bioinformatics and phylogenetic approaches to improve our understanding of the structural evolution of this important multigenic protein family. RESULTS: Organizing the I. ricinus single Kunitz-domain peptides based on their cysteine motif allowed us to specify a putative target and to relate this target specificity to Illumina transcript reads during tick feeding. We observe that several of these Kunitz peptide groups vary in their translated amino acid sequence, secondary structure, antigenicity, and intrinsic disorder, and that the majority of these groups are subject to a purifying (negative) selection. We finalize by describing the evolution and emergence of these Kunitz peptides. The overall interpretation of our analyses discloses a rapidly emerging Kunitz group with a distinct disulfide bond pattern from the I. ricinus salivary gland transcriptome. CONCLUSIONS: We propose a model to explain the structural and functional evolution of tick salivary Kunitz peptides that we call target-oriented evolution. Our study reveals that combining analytical approaches (transcriptomes, computational, bioinformatics and phylogenetics) improves our understanding of the biological functions of important salivary gland mediators during tick feeding.

Autonomous bacterial localization and gene expression based on nearby cell receptor density.

Escherichia coli were genetically modified to enable programmed motility, sensing, and actuation based on the density of features on nearby surfaces. Then, based on calculated feature density, these cells expressed marker proteins to indicate phenotypic response. Specifically, site-specific synthesis of bacterial quorum sensing autoinducer-2 (AI-2) is used to initiate and recruit motile cells. In our model system, we rewired E. coli's AI-2 signaling pathway to direct bacteria to a squamous cancer cell line of head and neck (SCCHN), where they initiate synthesis of a reporter (drug surrogate) based on a threshold density of epidermal growth factor receptor (EGFR). This represents a new type of controller for targeted drug delivery as actuation (synthesis and delivery) depends on a receptor density marking the diseased cell. The ability to survey local surfaces and initiate gene expression based on feature density represents a new area-based switch in synthetic biology that will find use beyond the proposed cancer model here.

Are ticks venomous animals?

INTRODUCTION: As an ecological adaptation venoms have evolved independently in several species of Metazoa. As haematophagous arthropods ticks are mainly considered as ectoparasites due to directly feeding on the skin of animal hosts. Ticks are of major importance since they serve as vectors for several diseases affecting humans and livestock animals. Ticks are rarely considered as venomous animals despite that tick saliva contains several protein families present in venomous taxa and that many Ixodida genera can induce paralysis and other types of toxicoses. Tick saliva was previously proposed as a special kind of venom since tick venom is used for blood feeding that counteracts host defense mechanisms. As a result, the present study provides evidence to reconsider the venomous properties of tick saliva. RESULTS: Based on our extensive literature mining and in silico research, we demonstrate that ticks share several similarities with other venomous taxa. Many tick salivary protein families and their previously described functions are homologous to proteins found in scorpion, spider, snake, platypus and bee venoms. This infers that there is a structural and functional convergence between several molecular components in tick saliva and the venoms from other recognized venomous taxa. We also highlight the fact that the immune response against tick saliva and venoms (from recognized venomous taxa) are both dominated by an allergic immunity background. Furthermore, by comparing the major molecular components of human saliva, as an example of a non-venomous animal, with that of ticks we find evidence that ticks resemble more venomous than non-venomous animals. Finally, we introduce our considerations regarding the evolution of venoms in Arachnida. CONCLUSIONS: Taking into account the composition of tick saliva, the venomous functions that ticks have while interacting with their hosts, and the distinguishable differences between human (non-venomous) and tick salivary proteins, we consider that ticks should be referred to as venomous ectoparasites.

Francisella DnaK inhibits tissue-nonspecific alkaline phosphatase.

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.

Dual SIFamide receptors in Ixodes salivary glands.

Salivary glands are vital to tick feeding success and also play a crucial role in tick-borne pathogen transmission. In previous studies of Ixodes scapularis salivary glands, we demonstrated that saliva-producing type II and III acini are innervated by neuropeptidergic axons which release different classes of neuropeptides via their terminals (Simo et al., 2009b, 2013). Among these, the neuropeptide SIFamide-along with its cognate receptor-were postulated to control the basally located acinar valve via basal epithelial and myoepithelial cells (Vancová et al., 2019). Here, we functionally characterized a second SIFamide receptor (SIFa_R2) from the I. scapularis genome and proved that it senses a low nanomolar level of its corresponding ligand. Insect SIFamide paralogs, SMYamides, also activated the receptor but less effectively compared to SIFamide. Bioinformatic and molecular dynamic analyses suggested that I. scapularis SIFamide receptors are class A GPCRs where the peptide amidated carboxy-terminus is oriented within the receptor binding cavity. The receptor was found to be expressed in Ixodes ricinus salivary glands, synganglia, midguts, trachea, and ovaries, but not in Malpighian tubules. Investigation of the temporal expression patterns suggests that the receptor transcript is highly expressed in unfed I. ricinus female salivary glands and then decreases during feeding. In synganglia, a significant transcript increase was detected in replete ticks. In salivary gland acini, an antibody targeting the SIFa_R2 recognized basal epithelial cells, myoepithelial cells, and basal granular cells in close proximity to the SIFamide-releasing axon terminals. Immunoreactivity was also detected in specific neurons distributed throughout various I. ricinus synganglion locations. The current findings, alongside previous reports from our group, indicate that the neuropeptide SIFamide acts via two different receptors that regulate distinct or common cell types in the basal region of type II and III acini in I. ricinus salivary glands. Our study investigates the peptidergic regulation of the I. ricinus salivary gland in detail, emphasizing the complexity of this system.

Pathway engineering via quorum sensing and sRNA riboregulators-interconnected networks and controllers.

The advent of genetic engineering has elevated our level of comprehension of cellular processes and functions. A natural progression of these findings is determining not only how these processes function within individual cells but also within a community. Bacterial cells monitor the conditions and microorganisms in their vicinity by producing, releasing and sensing chemical-signaling molecules. When a specific cell-density threshold is reached, a quorum is perceived, gene expression profiles are altered and the community orchestrates activities that are more effective en masse. This communication mechanism, in the language of autoinducers (AI), is referred to as quorum sensing (QS). It has become increasingly evident that while scientists attempt to decipher the intricacies of cellular communication and quorum sensing networks, we must remain conscious of the broader context of how a cell may identify itself in the environment and how this also impacts QS. Importantly, these phenomena span time and length scales by several orders in magnitude. Though the revelation of small RNAs, as both sensing and regulatory elements participating in the quorum sensing cascade, has connected new pieces of the puzzle, it has also added a new tier of uncertainty. The complexity of quorum sensing networks makes resolution of its diverse mechanisms difficult. The ability to design simpler networks with defined, more predictable or even "modular" elements will help elucidate these actions. Because it embraces innovative concepts of biological design accommodating the many length and time scales at play, synthetic biology serves as one of the most promising platforms for describing QS phenomena as well as enabling novel implementation strategies for biotechnological application.

LuxS coexpression enhances yields of recombinant proteins in Escherichia coli in part through posttranscriptional control of GroEL.

Cell-to-cell communication, or quorum sensing (QS), enables cell density-dependent regulation of bacterial gene expression which can be exploited for the autonomous-signal-guided expression of recombinant proteins (C. Y. Tsao, S. Hooshangi, H. C. Wu, J. J. Valdes, and W. E. Bentley, Metab. Eng. 12:291-297, 2010). Earlier observations that the metabolic potential of Escherichia coli is conveyed via the QS signaling molecule autoinducer-2 (AI-2) suggested that the capacity for protein synthesis could also be affected by AI-2 signaling (M. P. DeLisa, J. J. Valdes, and W. E. Bentley, J. Bacteriol. 183:2918-2928, 2001). In this work, we found that simply adding conditioned medium containing high levels of AI-2 at the same time as inducing the synthesis of recombinant proteins doubled the yield of active product. We have hypothesized that AI-2 signaling "conditions" cells as a natural consequence of cell-to-cell communication and that this could tweak the signal transduction cascade to alter the protein synthesis landscape. We inserted luxS (AI-2 synthase) into vectors which cosynthesized proteins of interest (organophosphorus hydrolase [OPH], chloramphenicol acetyltransferase [CAT], or UV-variant green fluorescent protein [GFPuv]) and evaluated the protein expression in luxS-deficient hosts. In this way, we altered the level of luxS in the cells in order to "tune" the synthesis of AI-2. We found conditions in which the protein yield was dramatically increased. Further studies demonstrated coincident upregulation of the chaperone GroEL, which may have facilitated higher yields and is shown for the first time to be positively regulated at the posttranscriptional level by AI-2. This report is the first to demonstrate that the protein synthesis capacity of E. coli can be altered by rewiring quorum sensing circuitry.

Enlisting the Ixodes scapularis Embryonic ISE6 Cell Line to Investigate the Neuronal Basis of Tick-Pathogen Interactions.

Neuropeptides are small signaling molecules expressed in the tick central nervous system, i.e., the synganglion. The neuronal-like Ixodes scapularis embryonic cell line, ISE6, is an effective tool frequently used for examining tick-pathogen interactions. We detected 37 neuropeptide transcripts in the I. scapularis ISE6 cell line using in silico methods, and six of these neuropeptide genes were used for experimental validation. Among these six neuropeptide genes, the tachykinin-related peptide (TRP) of ISE6 cells varied in transcript expression depending on the infection strain of the tick-borne pathogen, Anaplasma phagocytophilum. The immunocytochemistry of TRP revealed cytoplasmic expression in a prominent ISE6 cell subpopulation. The presence of TRP was also confirmed in A. phagocytophilum-infected ISE6 cells. The in situ hybridization and immunohistochemistry of TRP of I. scapularis synganglion revealed expression in distinct neuronal cells. In addition, TRP immunoreaction was detected in axons exiting the synganglion via peripheral nerves as well as in hemal nerve-associated lateral segmental organs. The characterization of a complete Ixodes neuropeptidome in ISE6 cells may serve as an effective in vitro tool to study how tick-borne pathogens interact with synganglion components that are vital to tick physiology. Therefore, our current study is a potential stepping stone for in vivo experiments to further examine the neuronal basis of tick-pathogen interactions.

Tick defensin γ-core reduces Fusarium graminearum growth and abrogates mycotoxins production with high efficiency.

Fusarium graminearum is a major fungal pathogen affecting crops of worldwide importance. F. graminearum produces type B trichothecene mycotoxins (TCTB), which are not fully eliminated during food and feed processing. Therefore, the best way to minimize TCTB contamination is to develop prevention strategies. Herein we show that treatment with the reduced form of the γ-core of the tick defensin DefMT3, referred to as TickCore3 (TC3), decreases F. graminearum growth and abrogates TCTB production. The oxidized form of TC3 loses antifungal activity, but retains anti-mycotoxin activity. Molecular dynamics show that TC3 is recruited by specific membrane phospholipids in F. graminearum and that membrane binding of the oxidized form of TC3 is unstable. Capping each of the three cysteine residues of TC3 with methyl groups reduces its inhibitory efficacy. Substitutions of the positively-charged residues lysine (Lys) 6 or arginine 7 by threonine had the highest and the lesser impact, respectively, on the anti-mycotoxin activity of TC3. We conclude that the binding of linear TC3 to F. graminearum membrane phospholipids is required for the antifungal activity of the reduced peptide. Besides, Lys6 appears essential for the anti-mycotoxin activity of the reduced peptide. Our results provide foundation for developing novel and environment-friendly strategies for controlling F. graminearum.

Autonomous induction of recombinant proteins by minimally rewiring native quorum sensing regulon of E. coli.

Quorum sensing (QS) enables an individual bacterium's metabolic state to be communicated to and ultimately control the phenotype of an emerging population. Harnessing the hierarchical nature of this signal transduction process may enable the exploitation of individual cell characteristics to direct or "program" entire populations of cells. We re-engineered the native QS regulon so that individual cell signals (autoinducers) are used to guide high level expression of recombinant proteins in E. coli populations. Specifically, the autoinducer-2 (AI-2) QS signal initiates and guides the overexpression of green fluorescent protein (GFP), chloramphenicol acetyl transferase (CAT) and beta-galactosidase (LacZ). The new process requires no supervision or input (e.g., sampling for optical density measurement, inducer addition, or medium exchange) and represents a low-cost, high-yield platform for recombinant protein production. Moreover, rewiring a native signal transduction circuit exemplifies an emerging class of metabolic engineering approaches that target regulatory functions.

SARS-CoV-2, Early Entry Events.

Viruses are obligate intracellular parasites, and host cell entry is the first step in the viral life cycle. The SARS-CoV-2 (COVID-19) entry process into susceptible host tissue cells is complex requiring (1) attachment of the virus via the conserved spike (S) protein receptor-binding motif (RBM) to the host cell angiotensin-converting-enzyme 2 (ACE2) receptor, (2) S protein proteolytic processing, and (3) membrane fusion. Spike protein processing occurs at two cleavage sites, i.e., S1/S2 and S2'. Cleavage at the S1/S2 and S2' sites ultimately gives rise to generation of competent fusion elements important in the merging of the host cell and viral membranes. Following cleavage, shedding of the S1 crown results in significant conformational changes and fusion peptide repositioning for target membrane insertion and fusion. Identification of specific protease involvement has been difficult due to the many cell types used and studied. However, it appears that S protein proteolytic cleavage is dependent on (1) furin and (2) serine protease transmembrane protease serine 2 proteases acting in tandem. Although at present not clear, increased SARS-CoV-2 S receptor-binding motif binding affinity and replication efficiency may in part account for observed differences in infectivity. Cleavage of the ACE2 receptor appears to be yet another layer of complexity in addition to forfeiture and/or alteration of ACE2 function which plays an important role in cardiovascular and immune function.